Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 2, pp. 241-256 © Pleiades Publishing, Ltd., 2024.

241

A Redox-Regulated, Heterodimeric

NADH:cinnamate Reductase in Vibrio ruber

Yulia V. Bertsova

1,a

, Marina V. Serebryakova

1,b

, Victor A. Anashkin

1,c

Alexander A. Baykov

1,d

, and Alexander V. Bogachev

1,e

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119234 Moscow, Russia

e-mail: bertsova@belozersky.msu.ru

e-mail: mserebr@mail.ru

e-mail: victor_anashkin@belozersky.msu.ru

e-mail: baykov@belozersky.msu.ru

e-mail: bogachev@belozersky.msu.ru

Received June 24, 2023

Revised October 9, 2023

Accepted October 10, 2023

Abstract— Genes of putative reductases of α,β-unsaturated carboxylic acids are abundant among anaerobic and

facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encod-

ed proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the facultatively anaerobic ma-

rine bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate

reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists of the

sjn56021-encoded subunit CrdB (NADH:flavin, FAD binding2, and FMN bind domains) and an additional subunit

CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 predic-

tion). Each domain contains a flavin group (three FMNs and one FAD in total), one of the FMN groups being linked

covalently by the flavin transferase. Crd readily reduces cinnamate, p-coumarate, caffeate, and ferulate under

anaerobic conditions with NADH or methyl viologen as the electron donor, is moderately active against acrylate

and practically inactive against urocanate and fumarate. Cinnamates induced Crd synthesis in V. ruber cells grown

aerobically or anaerobically. The Crd-catalyzed reduction started by NADH demonstrated a time lag of several

minutes, suggesting a redox regulation of the enzyme activity. The oxidized enzyme is inactive, which apparently

prevents production of reactive oxygen species under aerobic conditions. Our findings identify Crd as a regulated

NADH-dependent cinnamate reductase, apparently protecting V.ruber from (hydroxy)cinnamate poisoning.

DOI: 10.1134/S0006297924020056

Keywords: anaerobic respiration, cinnamic acid, caffeic acid, enzyme regulation, reactive oxygen species,

rhizosphere, Vibrio

Abbreviations: aCrd, non-flavinylated Crd protein containing only non-covalently bound flavins; fCrd, flavinylated Crd

protein; *FMN,covalently bound FMN residue; MS,mass spectrometry, MV, methyl viologen, m/z, mass-to-charge ratio;

MD,molecular dynamics; ROS,reactive oxygen species.

* To whom correspondence should be addressed.

INTRODUCTION

NADH:2-enoate reductases (EC 1.3.1.31) capable

of reducing α,β-unsaturated carboxylic acids, such as

fumaric, cinnamic, and acrylic acids, are abundant

among anaerobic and facultatively anaerobic micro-

organisms [1-4]. All known 2-enoate reductases are

formed by single polypeptides but are divided into two

non-homologous groups according to their variable

domain composition. The three-domain reductases

invariably contain a FAD binding 2 domain (PfamID:

PF00890), which has a noncovalently bound FAD

BERTSOVA et al.242

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Fig. 1. Bioinformatic description of V.ruber Crd and its genes. a)The domain composition of the polypeptides CrdA and CrdB

forming the Crd heterodimer. The NADH:flavin, FMN bind, and FAD binding2 domains (residues 1-180, 190-275, and 280-800, re-

spectively) are indicated by different colors. Domain names are taken from the Pfam database. Putative redox-active prosthetic

groups are shown inside the boxes. The asterisk denotes the covalently bound flavin. The truncated versions of Crd obtained in

this study are denoted as d1 and d3. b)Sequence alignment of the fumarate reductase from K. pneumoniae (Frd, UniProt acces-

sion number B5XRB0), acrylate reductase from V. harveyi (Ard, P0DW92), and CrdB protein from V. ruber (GenBank accession

number SJN56021) with Clustal [42]. The three parts of the alignment shown contain the amino acid residues involved in fuma-

rate C1- and C4-carboxylate binding in Frd (marked in blue and green, respectively), the proton transfer to fumarate (marked

in yellow) in Frd, and covalent bonding with FMN in all shown proteins (marked in red). c)The arrangement of the crd-associ-

ated genes in a V. ruber chromosome. ApbE, putative FAD:protein FMN transferase; CrdR, putative transcription regulator.

prosthetic group and is the place of carbonic acid re-

duction. NADH is oxidized in OYE-like (PF00724) or

FAD binding6 (PF00970) domain having noncovalently

bound FMN or FAD, respectively [2,3]. The third do-

main (commonly FMN bind; PF04205) mediates elec-

tron transfer between the above domains via a flavin

group (FMN) which is covalently bound by a phosphoe-

ster bond [4-6].

Many bacteria of the class Clostridia have a two-

domain 2-enoate reductase [1, 7], formed by OYE-like

and Pyr redox2 (PF07992) domains. The reductases of

this group contain a [4Fe-4S] cluster and noncovalent-

ly bound FAD and FMN in a 1 : 1 : 1 ratio as prosthetic

groups [8, 9]. In saccharolytic clostridia, two-domain

2-enoate reductases convert a broad range of 2-enoates,

whereas the enzymes from proteolytic clostridia are

highly specific and convert only cinnamate and its

derivatives [10, 11]. All known 2-enoate reductases do

not appear to be regulated at activity level, except by

substrates and products.

The genome of the red facultatively anaerobic ma-

rine bacterium Vibrio ruber [12] encodes a polypeptide

formed by 806 amino acid residues and annotated as

urocanate reductase in GenBank (SJN56021) and in

UniProt (A0A1R4LHH9). This polypeptide, which we

designate as CrdB, based on the substrate specificity

of its containing enzyme (see below), encompasses the

abovementioned FAD binding2 and FMN bind domains

(Fig. 1a). Five putative substrate-binding residues, iden-

tified in the FAD binding 2 domains of CrdB and acry-

late reductase of Vibrio harveyi by comparison with

fumarate reductase [4, 13, 14] (Fig.1b), are identical or

very similar, suggesting that CrdB is an acrylate reduc-

tase. The FMN bind domain of CrdB, like its counter-

CINNAMATE REDUCTASE OF V. ruber 243

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

parts in known 2-enoate reductases, contains the motif

DALSGAS

257

recognized by flavin transferase for cova-

lent FMN attachment to the serine residue [15]. Note-

worthy, the gene apbE encoding the flavin transferase

(GenBankID: SJN56016) locates nearby to crdB in one

of the two V. ruber chromosomes (Fig. 1c). Altogether,

these characteristics of CrdB classify it as a three-do-

main 2-enoate reductase with a likely acrylate reduc-

tase activity.

However, the CrdB protein differs from estab-

lished three-domain 2-enoate reductases by its third,

NADH:flavin domain (PF03358) replacing the NADH-

oxidizing domains OYE-like or FAD binding 6 (Fig. 1a).

Furthermore, the crd-operon of V. ruber contains, up-

stream of the crdB gene, the gene crdA encoding a pro-

tein (SJN56019) (Fig.1c) formed by a single NADH:flavin

domain that is homologous (51% identity, 65% similar-

ity) to the similar domain of CrdB. As NADH:flavin do-

mains can dimerize [16], this observation raised an in-

triguing possibility of Crd functioning as a CrdA/CrdB

heterodimer.

Keeping in mind the difficulty in the theoretical

prediction of the enzymatic activities and substrate

specificities of NADH:2-enoate reductases, we have

produced the V. ruber Crd protein in Escherichia coli

cells and characterized the isolated protein. The data

reported below identify Crd as a regulated NADH-de-

pendent reductase active against cinnamic acid and its

various derivatives.

MATERIALS AND METHODS

Bacterial strains and growth conditions. V. ru-

ber DSM16370 was obtained from the Leibniz Institute

collection of microorganisms and cell cultures (DSMZ).

The cells were grown aerobically or anaerobically at

28°C in a mineral medium containing 20 g/liter NaCl,

0.75 g/liter KCl, 1.2 g/liter MgSO

×7(H

O), 0.5 g/liter NH

Cl,

0.5 mM Na

HPO

, 2 g/liter sucrose, 0.5 g/liter yeast ex-

tract, and 50 mM Tris-HCl (pH 8.0). When required, the

medium was supplemented with 2-10 mM acrylate, cin-

namate, p-coumarate, caffeate, or ferulate. Cleared cell

lysate was prepared as described elsewhere [17] and

assayed for MV:cinnamate reductase activity. E. coli

cells were grown at 37°C in LB. Where indicated, the

LB medium was supplemented with 20 µg/ml chloram-

phenicol and(or) 100 µg/ml ampicillin.

Construction of expression vectors. The ex-

pression vector for the CrdAB protein with C-terminal

6×His-tagged CrdB was constructed by amplifying the

crd-operon from the genomic V. ruber DNA. A high-

fidelity Tersus polymerase (Evrogen, Russia) and the

primers V_ruber_DIR/V_ruber_REV were used (prim-

er sequences are listed in Table S1 in the Online Re-

sourse1). The resulting 3276-bp fragment was cloned

into the pBAD-TOPO vector (Invitrogen, USA), yielding

the pTB_CRD4 plasmid. The plasmid was transformed

into E.coli/pΔhis3 [18] or BL21 strains.

The expression vector for the d1 fragment (full-

size CrdA and residues 1-184 of CrdB) was construct-

ed by amplifying the corresponding DNA fragment of

the crd-operon by PCR with a Tersus PCR kit and the

primers V_ruber_DIR/VR_P1R using V. ruber genomic

DNA as the template. The resulting 1401-bp fragment

was cloned into the pBAD-TOPO vector, yielding the

pTB_d1CRD17 plasmid. To construct an expression

vector for the d3 fragment (residues 281-806) of CrdB,

the 1688-bp fragment was amplified by PCR with

the VR_3P2_NdeD/Xho_rhod_rev primer pair and the

pTB_CRD4 plasmid as the template. The amplified frag-

ment was cloned into the pSCodon vector (Delphi Ge-

netics, Belgium) using the NdeI/XhoI sites, resulting

in the plasmid pSC_d3CRD2. The pTB_d1CRD17 and

pSC_d3CRD2 plasmids were transformed into E. coli

BL21 strain.

Crd production. The 6×His-tagged Crd proteins

with or without covalently bound FMN (fCrd and aCrd,

respectively) and the d1 and d3 fragments of Crd were

produced in E. coli BL21 or pΔhis3 cells and purified

using metal chelate chromatography as described pre-

viously [5]. The extinction coefficients ε

450nm

for fCrd,

aCrd, d1 fragment, and d3 fragment determined after

SDS treatment [4] were 42, 31, 22, and 11 mM

–1

respectively.

Analysis of flavins. Non-covalently bound flavins

were extracted from Crd preparations by trifluoroace-

tic acid (TFA) and separated by HPLC [5]. SDS-PAGE

was performed using 12.5% (w/v) polyacrylamide gels

[19]. The gels were stained for protein with PageBlue

solution (Fermentas, Lithuania). Covalently bound fla-

vins were detected by scanning unstained gels with

a Typhoon

FLA 9500 laser scanner (GE Healthcare,

USA) with excitation at 473 nm and the detection of

emission using the SYBR Green II protocol according

tothe manufacturer’s recommendations.

For mass-spectral measurements, gel pieces of

approximately 2 mm

were excised from the protein

bands of the Coomassie-stained gel and destained

by incubating in two 0.1-ml volumes of 40% (by vol-

ume) acetonitrile solution containing 20 mM NH

HCO

pH 7.5, dehydrated with 0.2 ml of 100% acetonitrile

and rehydrated with 5 μl of the digestion solution con-

taining 15 µg/ml sequencing-grade trypsin (Promega,

USA) in 20 mM aqueous solution of NH

HCO

, pH 7.5.

Digestion was carried out at 37°C for 1 h. The result-

ing peptides were extracted with 5 μl 30% acetoni-

trile solution containing 0.5% TFA. A 1-μl aliquot of

the in-gel tryptic digest extract was mixed with 0.5μl

of 2,5-dihydroxybenzoic acid solution (40 mg/ml) in

30% acetonitrile containing 0.5% TFA and left to dry on

a stainless-steel target plate. MALDI-TOF MS analysis

BERTSOVA et al.244

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

was performed on an UltrafleXetreme MALDI-TOF-

TOF mass spectrometer (Bruker Daltonik, Germany).

The MH

molecular ions were measured in a reflector

mode; the accuracy of monoisotopic mass peak mea-

surement was within 30ppm. Spectra of fragmentation

were obtained in a LIFT mode; the accuracy of daugh-

ter ion measurement was within 1 Da. Mass-spectra

were processed with the FlexAnalysis 3.2 software

(Bruker Daltonik). Protein identification was carried

out by MS+MS/MS ion search, using Mascot software

version2.3.02 (Matrix Science, USA), through the Home

NCBI Protein Database. One each of missed cleavage,

Met oxidation, Cys-propionamide, and Ser(Thr)-FMN

were permitted. Protein scores greater than 94 were

considered significant (p<0.05).

Enzymatic activities. NADH- and methyl viologen

(MV)-supported activities of Crd preparations were de-

termined at 25°C by following NADH or MV oxidation

spectrophotometrically at 340 or 606 nm, respectively,

with a Hitachi-557 spectrophotometer [3]. The assay

medium contained 1 mM MV, 0.03-1 mM electron ac-

ceptor, and 100 mM Tris-HCl (pH 8.0) or 120 μM NADH,

0.05-1 mM electron acceptor, 10 mM glucose, 5 U/ml

glucose oxidase, 5 U/ml catalase, and 100 mM Mes-KOH

(pH 6.5) in a completely filled and sealed 3.2-ml cu-

vette. MV was pre-reduced with sodium dithionite until

the absorbance at 606 nm of approximately 1.5 was ob-

tained, which corresponds to the formation of ~100 μM

reduced MV. Unless otherwise noted, Crd or its variant

was preincubated with NADH or MV for 5min before

the reaction was started by adding an electron accep-

tor. The NADH-supported reaction was also measured

under aerobic conditions without glucose oxidase and

catalase. Crd activities were calculated using the ex-

perimentally verified NADH:acceptor and MV:acceptor

stoichiometries of 1:1 and 2:1, respectively.

The assay medium for measuring FMNH

:cinna-

mate reductase activity contained 100 μM FMN, 1 mM

cinnamate, and 100 mM Tris-HCl (pH 8.0). FMN was

pre- reduced with sodium dithionite until the absor-

bance at 450 nm decreased to a value of ~0.3, which

corresponds to formation of ~70 μM reduced FMN.

FMNH

oxidation was followed spectrophotometrically

at 450nm.

Phenylpropionate dehydrogenase activity was as-

sayed by measuring the reduction of 2,6-dichloropheno-

lindophenol (DCPIP, ε

600

 = 22 mM

–1

) at 600 nm[20].

The assay mixture contained 2 mM Tris-phenylpropi-

onate, 2 mM phenazine methosulphate, 25 μM DCPIP,

and 100mM Mes-KOH (pH6.5).

The Michaelis–Menten parameters of MV-sup-

ported acrylate reduction by Crd were estimated from

duplicate measurements of the initial rates of MV oxi-

dation using 0.03-10 mM acrylate and 40 nM Crd con-

centrations. For other electron acceptors, rates were

obtained from the integral kinetics of their reduction

until its completion, as monitored by absorbance

at 606 nm. Rates were estimated at 100 time points

along the progress curve as the slopes of the tangents

(-d[MV]/dt) using MATLAB (The MathWorks, Inc.).

Theresidual electron acceptor concentration was cal-

culated at each point from A

606

using the MV : accep-

tor stoichiometry of 2 : 1 and assuming that the lim-

iting value of A

606

corresponds to 100% conversion of

the electron acceptor. The Michaelis–Menten equation

was fitted to the rate data using non-linear regression

analysis.

Identification of the product of cinnamate

reduction. Cinnamate reduction was performed in

3.2 ml of the medium containing 100 μM cinnamate,

10 mM sodium dithionite, 50 μM MV, and 100 mM

Tris-HCl (pH 8.0). The reaction was initiated by add-

ing 0.5 μM Crd and terminated after 30min by adding

5% (vol/vol) HClO

. In the control experiment, HClO

was added before Crd. Precipitated protein was re-

moved by centrifugation, and the substrate and prod-

ucts were extracted from the samples with diethyl

ether (2 × 1.5 ml). The ether was evaporated under a

stream of air, and the residue was dissolved in 1 ml

of 25 mM potassium phosphate (pH 6.5) (medium A).

The samples were separated by HPLC on a ProntoSil-

120-5-C18 AQ column using a Milichrom A-02 chro-

matograph (both from Econova, Russia). The column

was pre-equilibrated with medium A and eluted with a

linear gradient from 0% to 10% methanol in medium A

at a flow rate of 0.2 ml/min with UV detection at 210

and 258 nm.

Quantitative reverse transcription polymerase

chain reaction (RT-qPCR). RNA extraction from V.ruber

cells and cDNA synthesis were performed as described

previously [17]. RT-qPCR assays were performed with

qPCRmix-HS SYBR kit (Evrogen), using the cDNA prepa-

rations as templates and VR_69U19/VR_273L20 as

primer pair for crdB. 16S rRNA was used for data nor-

malization (the primer pair 16s FW/16s RV). Serial di-

lutions of V. ruber genomic DNA, which contains the

genes for CrdB and 16S rRNA in a 1 : 8 ratio, were used

for calibration.

Bioinformatics. The three-dimensional structure

of flavin-deficient Crd was predicted from the amino

acid sequences of CrdA and CrdB using AlphaFold2

(version 2.2.0) [21] through the ColabFold advanced

notebook [22]. Multimer model prediction was per-

formed using default parameter settings. The Ramach-

andran plot indicated that 96.7% residues have favor-

able dihedral angles in the modelled structure.

Ligands (flavins, NADH, cinnamate) were docked

in sequence into the obtained Crd structure using

AutoDock Vina [23] in the following sequence: *FMN,

FAD, FMN

, FMN

, NADH, cinnamate. Space cells rang-

ing from 12×12×12 to 30×30×30 Å

were selected for

docking in the appropriate regions of the protein based

CINNAMATE REDUCTASE OF V. ruber 245

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

on the known structures of homologous proteins.

Each docking experiment was performed in triplicate.

The derived Crd structure containing one FAD and

three FMN groups was finally equilibrated by mo-

lecular dynamics (MD) simulations for 150 ns with

AMBER 22 package [24] (https://ambermd.org/). Details

of the simulation are found in Electronic Supplementa-

ry Materials (Fig.S4 legend in the Online Resourse1).

RESULTS

Production and characterization of V. ruber Crd.

The CrdAB operon of V.ruber genomic DNA was ampli-

fied and cloned into an expression vector that added

a 6×His tag at the C-terminus of CrdB. The FMN bind

domain of CrdB harbors the sequence DALSGAS

257

similar to the flavinylation motif Dxx(s/t)gA(T/S) recog-

nized by flavin transferase for covalent attachment of

FMN via a threonine or serine hydroxyl [15]. To test

whether the predicted flavinylation really occurs, the

crd-operon genes were expressed in E. coli cells in the

presence or absence of the auxiliary pΔhis3 plasmid

[18] that encodes the flavin transferase ApbE capable

of flavinylating FMNbind domains in various proteins

[15]. The recombinant fCrd and aCrd proteins pro-

duced in the ApbE-containing and ApbE-lacking cells,

respectively, were isolated by metal affinity chroma-

tography. The final protein preparations were of intense

yellow color, and their visible spectra (Fig. 2a) were

characteristic of flavoproteins.

SDS-PAGE of both Crd preparations revealed one

major and three-four minor protein bands (Fig. 2b,

left image). Bands 1 and 3 were identified by the

MALDI-MS analysis after trypsin digestion as V. ruber

CrdB (sequence coverage of 79%) and CrdA (sequence

coverage of 95%), respectively. Band 2 belonged to

E. coli peptidyl-prolyl cis-trans isomerase SlyD, which

exhibits high intrinsic affinity to Ni-agarose and is a

common contamination of the 6×His-tagged proteins

isolated from E. coli [25]. Band 1 derived from fCrd

fluoresced when illuminated at 473nm, indicating co-

valently bound flavin (Fig. 2b, right image). No bound

flavin was detected by this method in aCrd. These find-

ings provide convincing evidence for ApbE-mediated

covalent flavinylation of CrdB. It should be noted that

the non-covalently bound flavins, evidently present in

both Crd preparations (Fig.2a) are removed from the

proteins during SDS-PAGE.

The CrdB flavinylation site was identified by

MALDI-MS and MS/MS after trypsinolysis. The pep-

tide I

242

IEGQTLNVDALSGASETSHAVIDGVAK

269

contain-

ing the underlined flavinylation motif should have

demonstrated the monoisotopic MH

masses of 2795.3

and 3233.5 in the non-flavinylated and flavinylated

forms, respectively. Both predicted signals were indeed

Fig. 2. Characterization of the Crd preparations. a)Electron-

ic absorption spectra of the full-length and truncated (d1 and

d2) Crd preparations at 10 μM concentrations. Full-length

Crd was produced in ApbE-containing or ApbE-lacking E. coli

cells (fCrd and aCrd, respectively). b) SDS-PAGE of fCrd and

aCrd. The gel was stained with Coomassie Blue (left image)

or scanned under illumination at 473 nm without staining

(right image). The protein load was 5 µg per lane. Bars with

numbers on the left side denote the positions and molecular

masses of marker proteins. The protein bands identified by

MALDI-MS analysis are marked by numbers. c)HPLC separa-

tion of non-covalently bound flavins in Crd and its truncated

variants. The retention volumes for authentic FAD, FMN, and

riboflavin(Rf) are indicated by arrows.

BERTSOVA et al.246

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

observed on the mass-spectrum of the tryptic digest of

fCrdB. The presence of the former signal may reflect

either incomplete modification or partial loss of the

flavin group during SDS-PAGE and trypsin proteolysis.

No other candidates for flavinylated peptides were ob-

served in the tryptic digest of CrdB and CrdA by a pep-

tide fingerprint Mascot search.

The MS/MS analysis of the peptide with m/z of

3233.5 (Fig.S1 in the Online Resourse1) indicated that

the main signals in the spectrum of fragmentation (m/z

of 2794 and 2776) resulted from FMN loss in the full

or dehydrated form, respectively, depending on which

of the two covalent bonds, C–O or O–P, connecting the

FMN moiety to the serine residue is broken. Greater

intensity of the signal with m/z of 2776 indicated that

the former bond is broken with greater propensity,

resulting in serine dehydration [26]. Less intense but

still reliable peaks in the spectrum starting from the

fragment with m/z = 2776 also matched a series of the

y-ions generated by double breaks during the fragmen-

tation (Fig. S1, red letters, see the Online Resourse 1).

Here, mass difference for the Ser257 position (69 Da)

corresponded to the dehydrated serine, demonstrat-

ing that fCrdB produced in ApbE-containing cells

harbors an FMN residue covalently bonded to Ser257

of the predicted flavinylation motif in the FMN bind

domain.

The non-covalently bound flavins were extracted

from Crd and separated by HPLC (Fig. 2c). Both the fCrd

and aCrd preparations were found to contain non-co-

valently bound FAD and FMN at a ratio of approxi-

mately 1:2.

To determine their domain localization, we pre-

pared the genetic constructs encoding separately two

NADH:flavin domains of CrdA and CrdB (d1 in Fig. 1a)

or the FAD binding 2 domain of CrdB (d3 in Fig. 1a).

Theisolated corresponding proteins exhibited the spec-

tra characteristic of flavoproteins (Fig.2a). Theflavins

present in them were again extracted and identified

by HPLC (Fig.2c). The non-covalently bound FMN was

exclusively localized in the d1 fragment (NADH:flavin

domains) whereas FAD was found in the d3 fragment

(FAD binding 2 domain). The evident corollary is that

CrdAB contains two non-covalently bound FMN mole-

cules in the oxidoreductase NADH:flavin domains, one

non-covalently bound FAD molecule in the FAD bind-

ing2 domain, and one covalently bound FMN residue

in the FMNbind domain.

The specificity of Crd for naturally occurring

α,β-unsaturated carbonic acids. The similarity of the

putative substrate-binding residues in the FAD bind-

ing 2 domains of Crd and the acrylate reductase Ard

of V. harveyi (Fig. 1b) raised the possibility that Crd

also possesses acrylate reductase activity. Indeed, the

Crd preparation obtained by crdAB-apbE coexpression

(fCrd) could reduce acrylate using reduced methyl

viologen (MV) as the electron donor, but at a much

lower rate (1.3 s

–1

) compared with that of a specif-

ic acrylate reductase (19 s

–1

 [4]). Conversely, the K

value was much greater for fCrd (770 versus 16 µM),

making its catalytic efficiency k

cat

640 times lower

(0.0019 µM

–1

) compared with the acrylate reductase

(1.2 µM

–1

 [4]). fCrd exhibited low but measurable ac-

tivity against fumarate but was virtually inactive with

methacrylate, crotonate, and urocanate (Table1).

In contrast, cinnamate and its hydroxy deriva-

tives were readily reduced by fCrd (k

cat

of 22-47 s

–1

) and

demonstrated low K

values of 2.2-7.1 µM (Table 1).

These findings classify Crd as a cinnamate reductase

that is also active with other natural α,β-unsaturated

cinnamate derivatives.

To determine the product of cinnamate reduction

by fCrd, we identify it using HPLC. The Crd-catalyzed

reaction was found to completely convert cinnamate

to phenylpropionate (Fig. 3, a and b). The identifica-

tion of the carbonic acids was supported by the ob-

servation that the peak heights on the elution profiles

monitored at 210 and 258nm were nearly equal for cin-

namate but differed approximately 40-fold for phenyl-

propionate, in accordance with the UV-spectra of these

compounds (Fig. 3c). These findings indicate that Crd

reduces the α,β double bond in (hydroxy)cinnamic ac-

ids to yield their saturated derivatives. fCrd activity

in the reverse reaction of phenylpropionate oxidation

with phenazine methasulfate and dichlorophenolin-

dophenol as electron acceptors was measurable but

quite low (0.1s

–1

Induction of Crd synthesis in V. ruber cells by

unsaturated carbonic acids. To determine whether

the found Crd substrates induce Crd synthesis in V.ru-

ber, cell growth was performed in their presence and

the MV:cinnamate reductase activity was measured in

cleared cell lysates. All cultivations were performed in

duplicate both in the absence and in the presence ofO

As Table 2 makes clear, cinnamate added at its

maximal concentration (10 mM) that did not yet sup-

press cell growth caused appreciable induction of Crd

synthesis. The effect was observed under both aero-

bic and anaerobic conditions but was maximal in the

former case. Parallel measurements indicated a more

than ten-fold increase in the gene crdB transcription

by cinnamate, providing further support for assigning

the MV:cinnamate reductase activity to Crd in V.ruber

cells.

The other α,β-unsaturated carbonic acids acting

as Crd substrates, except for acrylate, could induce Crd

synthesis under anaerobic conditions (Fig. 4). These

compounds were added at 2 mM concentrations be-

cause the cells did not grow anaerobically at higher

concentrations of acrylate and caffeate. The effects of

the carbonic acids on Crd induction thus correlated

with their ability to act as substrates (Table1).

CINNAMATE REDUCTASE OF V. ruber 247

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

Table 1. The reductase activity of fCrd against a series

of α,β-unsaturated carbonic acids measured under

anaerobic conditions with 100µM reduced MV as the

electron donor

Substrate

(acid)

Structure k

cat

–1

) K

(µM)

Caffeic

47 ± 2 2.2 ± 0.4

p-coumaric

45 ± 3 2.4 ± 0.5

Ferulic

26 ± 2 2.8 ± 0.5

Cinnamic

22 ± 1.5 7.1 ± 0.6

Acrylic

1.3 ± 0.2 770 ± 50

Fumaric 0.20 ± 0.03

Crotonic 0.06 ± 0.02

Urocanic 0.04 ± 0.01

Methacrylic 0.03 ± 0.01

Value of activity measured at 1mM substrate concentration.

Time-dependent activation of isolated Crd by

NADH. The presence of the NADH:flavin domains

in CdrAB (Fig. 1a) suggested that NADH is the natu-

ral donor of the redox equivalents for this enzyme.

As ferulate, caffeate, and p-coumarate absorb light at

340 nm, the NADH dehydrogenase activity was tested

with non-absorbing cinnamate as the electron accep-

tor. As the red trace in Fig.5a highlights, the reaction

initiated by cinnamate addition under anaerobic con-

ditions demonstrated a linear NADH oxidation curve

Fig. 3. HPLC identification of the product of cinnamate reduc-

tion by fCrd. Cinnamate (0.1mM) was reacted with 10mM so-

dium dithionite and 50µM MV in the presence of 0.5µM fCrd.

a)The reaction mixture before fCrd addition. b)Same after a

30-min incubation with fCrd. The elution was monitored at

210 (blue curve) and 258nm (red curve). The elution volumes

for authentic cinnamate and phenylpropionate are indicat-

ed by arrows. c) Electronic absorption spectra of cinnamate

and phenylpropionate in 10mM potassium phosphate buffer

(pH6.5).

Table 2. Correlation between the MV:cinnamate reductase activity and crdB transcription level in the V. ruber

cells cultivated in different conditions

Growth conditions Crd activity (nmol·min

–1

·mg

–1

) crdB mRNA/rRNA×10

–6

No cinnamate, no O

1.2 ± 0.4 2.1 ± 0.2

No cinnamate, +O

0.5 ± 0.2 0.8 ± 0.1

10 mM cinnamate, no O

86 ± 11 31 ± 4

10 mM cinnamate, +O

19 ± 3 25 ± 2

BERTSOVA et al.248

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

and a relatively high rate of 15 s

–1

. However, changing

the order of the additions affected the reaction time

course dramatically. For the reaction started by fCrd,

the progress curve demonstrated a profound lag in the

NADH oxidation (blue trace in Fig. 5a). Although the

initial slope was quite low, the final slope was similar

to that for the red trace described above and likely

corresponded to fully activated Crd. A similar time lag

was observed in the reaction started by NADH.

These findings indicate that fCrd is inactive as iso-

lated and requires preincubation with NADH for acti-

vation. That the length of the time lag of anaerobic cin-

namate reduction started by fCrd did not depend on

fCrd concentration (data not shown) indicated that the

activation is not associated with changes in the oligo-

meric structure. Furthermore, the red trace in Fig. 5a

demonstrates that the activation is much faster in the

absence of cinnamate than in its presence (blue curve).

The duration of Crd preincubation with NADH for the

red curve in Fig.5a was approximately 5min and this

time was thus sufficient for nearly complete activation

in the absence of the electron acceptor. Preincubation

with 5 mM dithionite for 5 min before starting the re-

action by the enzyme activated fCrd to the same level

(data not shown). These findings suggested that the ac-

tivation is likely caused by the reduction of a prosthetic

group in fCrd rather than by mere NADH binding to it.

For further analysis, the product formation curve

for the reaction started by Crd in Fig.5a (blue curve)

was converted into the first derivative (i.e., specific

activity) plot (Fig.5b), and its sigmoidal shape clearly

indicated that the activation process is not a one-step

reaction. Fitting experiments demonstrated a poor fit

of the kinetic scheme involving two consecutive steps

(red line) but a reasonably good fit for a scheme in-

volving three such steps (green line). In these fittings,

we assumed that the rate constants for each step are

identical because this relationship corresponds to

the largest possible time lag of the final product for-

mation. The fitted value of the rate constant was

0.060 ± 0.003 s

–1

for the three-step reaction. Three is

thus the minimal number of the steps involved in slow

Crd activation by NADH.

A similar but faster activation of Crd was ob-

served in the anaerobic acrylate reduction started by

the enzyme (Fig.5c). The maximal activity measured

with this substrate (3.5 s

–1

) was 23% of that with cin-

namate. For comparison, this activity was only 1.3 s

–1

with MV as the electron donor (6% of the activity with

cinnamate, Table1). A similar kinetic analysis indicated

that Crd activation by NADH in the presence of acry-

late involves at least two steps (Fig.S2 in the Online Re-

sourse 1), both with the rate constant of 0.27 ± 0.02 s

–1

We also demonstrated that Crd activation by NADH in

the absence of electron acceptors (Fig. S2, left panel,

squares, see the Online Resourse 1) was faster than

in their presence (Fig. S2, left panel, circles, see the

Online Resourse1; Fig.5b, circles). Asimilarly kinetic

analysis has revealed that the activation also involved

two consecutive steps with the average rate constant

of 0.75 ± 0.15 s

–1

If MV was used as the electron donor with various

substrates (Table 1), the progress curves were always

linear irrespective of the order of fCrd and substrate

additions. This result suggested that the activation/

deactivation of fCrd is somehow associated with its

NADH-binding NADH:flavin domains. This hypothesis

was supported by the observation that the anaerobic

NADH:FMN reduction by fCrd also demonstrated slow

activation if the reaction was started by the enzyme

(Fig.5d). The activation kinetics was more complex in

this case and demonstrated a poor fit of the consecu-

tive three-step model (Fig.S2, right panel in the Online

Resourse1).

Crd activated by preincubation with NADH re-

mained active until all NADH was consumed in the

enzymatic reaction, but slowly returned to the inac-

tive state afterwards. In the experiment illustrated

in Fig. 6a, the reaction was started by adding acry-

late to preactivated fCrd and proceeded linearly until

NADH exhaustion. A new portion of NADH restored

Crd activity to the level that depended on the time

interval t before the second NADH addition (Fig. 6b).

Thetime-courses became nonlinear at hight, indicat-

ing activation as in Fig.5c (blue trace) to a final rate

Fig. 4. MV:cinnamate reductase activity of the V. ruber cells

grown anaerobically in the presence of 2mM caffeate (caf),

p-coumarate (cou), ferulate (fer), cinnamate (cin), or acry-

late (acr). Error bars represent the standard deviations for

two cultivations.

CINNAMATE REDUCTASE OF V. ruber 249

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

similar to those in Fig.5c (red trace) and 6a (left trace).

These findings indicate that active fCrd is gradually de-

activated when depleted of NADH but time-dependent-

ly restores full activity after NADH addition. fCrd deac-

tivation was a first-order reaction with a half-time of

4.7min (Fig.6b).

Under aerobic conditions, the NADH-oxidizing ac-

tivity of Crd was extremely low and did not increase

with time (Fig.7, black (1) trace). This means that ox-

ygen prevents Crd conversion from inactive to active

state. This observation contrasts the data reported for

other NADH:2-enoate and NADH:flavin reductases,

which readily oxidize NADH with O

as the main elec-

tron acceptor [3, 4, 27]. To determine oxygen effect on

active Crd, the enzyme was preincubated with NADH

and allowed to convert excess cinnamate under anaer-

obic conditions before oxygen was added in the form

of H

(Fig.7, red (2) trace). In this experiment, lower

concentration of glucose oxidase and higher concen-

tration of catalase were used to maintain oxygen level

Fig. 5. The typical traces of fCrd-catalyzed NADH-dependent reactions under anaerobic conditions. a)Cinnamate-supported

NADH oxidation. The final reaction mixture contained 120 µM NADH, 50 µM cinnamate, and 30 nM fCrd. The reaction was start-

ed by either cinnamate (cin, red trace) or fCrd (blue trace). b)The time dependence of Crd activity derived from the time-course

of cinnamate reduction in the reaction started by Crd addition (blue curve on panela). Small circles show the activity values

determined as the slope of the tangent (-d[NADH]/dt) using Matlab (The Mathworks, Inc.) at 100 time points. The red and green

lines show the best fits for two- and three-step kinetic models, respectively. c)Acrylate-supported NADH oxidation. The final

reaction medium contained 120 µM NADH, 1 mM acrylate, and 150 nM fCrd. The reaction was started by acrylate (acr, red trace)

or fCrd (blue trace). d)NADH-supported FMN reduction. The final reaction medium contained 120 µM NADH and 50 μM FMN.

ThefCrd concentration was 15 nM (red trace, reaction started by FMN) or 60 nM (blue trace, reaction started by fCrd).

BERTSOVA et al.250

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

in the incubation mix. As Fig.7 highlights, the oxygen

pulse caused slow inactivation of Crd, followed by slow

reactivation to the initial level upon oxygen depletion

by glucose oxidase. The duration of the transient inacti-

vation phase decreased with increasing glucose oxidase

concentration (data not shown). These findings indicat-

ed that the reaction of molecular oxygen with Crd, re-

sponsible for its inactivation, is easily reversible, allow-

ing regulation of Crd activity by medium oxygen level.

Interestingly, NADPH demonstrated only low effi-

ciency as an electron donor compared with NADH. In

the corresponding experiment, fCrd preactivated by

incubation with 30µM NADH was allowed to convert

acrylate until all NADH was consumed and immediately

supplemented with 120µM NADPH. The measured rate

of NADPH oxidation was 35 times lower than the rate

of NADH oxidation. This makes NADH the most likely

physiological donor of the reducing equivalents for Crd.

Domain roles in the catalytic activities of Crd.

Crd is thus capable of catalyzing redox reactions be-

tween different natural and artificial donors and ac-

ceptors of electrons. To determine which Crd domains

are responsible for different reactions, we measured

the activities of the apo- and flavinylated forms of Crd

and its deletion variants. The MV:cinnamate reductase

activity was high with fCrd and much lower with aCrd

(no covalently bound FMN) and d3 (only FAD binding2

domain) (Table 3). These data indicated that, like in

Klebsiella pneumoniae NADH:fumarate reductase [3],

the FAD binding2 domain is required for reduction of

natural electron acceptor and the electron pathway

from MV to this domain involves the covalently bound

FMN of the FMN bind domain.

In contrast, high NADH:FMN reductase activity

was demonstrated not only by the full-size flavinylat-

ed enzyme (fCrd) but also by its apo-form and the d1

variant (only NADH:flavin domains). This reaction is

thus clearly catalyzed by the NADH:flavin domains.

Noteworthy, all the NADH:FMN reductase activities, in-

cluding that manifested by the d1 variant, developed

slowly in time when the reaction was started by NADH

but instantly when FMN was the last added component,

as in Fig.5d.

Full NADH:cinnamate reductase activity was man-

ifested by fCrd but not aCrd that lacks covalently

bound FMN. These findings emphasized the role of the

covalently bound FMN in electron transfer between

the NADH dehydrogenase part(d1) and the cinnamate

reductase part(d3) in Crd.

Fig. 6. Deactivation of Crd. a)Anaerobic NADH oxidation by

fCrd in the presence of 1 mM acrylate(acr). When the reac-

tion ended because of NADH consumption, its concentra-

tion was restored to the initial level (~50 µM) after a 1.5-min

pause. Conditions were as for Fig.5c. b)The initial activity of

NADH conversion measured for the second portion of NADH

as a function of the time interval t before its re-addition.

100% refers to the initial activity estimated from the left

curve in panel(a). The theoretical curve is for the first-order

reaction with a half-time of 4.7min.

Fig. 7. Transient inactivation of Crd by oxygen pulse during

NADH-supported cinnamate reduction. Trace 1) The reaction

started by fCrd and carried out under aerobic conditions

(no glucose oxidase and catalase added). The experimental

conditions were otherwise as for the red curve in Fig. 5a.

Trace 2) The reaction was started by 500 µM cinnamate and

carried out under anaerobic conditions until 500 μM H

(producing 250 µM molecular oxygen), were added at the

indicated time. The concentrations of glucose oxidase and

catalase in this particular experiment were 0.6 and 50U/ml,

respectively. The final reaction mixture contained 120 µM

NADH and 30nM fCrd in both experiments.

CINNAMATE REDUCTASE OF V. ruber 251

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

The separate FAD binding 2 domain (d3 variant)

demonstrated measurable activity (0.60 ± 0.05 s

–1

, data

not shown) in FMNH

:cinnamate reduction, raising the

possibility of catalyzing complete NADH:cinnamate

reductase reaction by a 1 : 1 mixture of d1 and d3 in

the presence of medium FMN. Such activity was in-

deed detected, and although its value, 3.2 ± 0.4 s

–1

(data

not shown), was less than for fCrd, it significantly ex-

ceeded the FMNH

:cinnamate reductase activity of d3.

This seemingly unexpected result could be, however,

explained by different conditions used for the two re-

actions. The assay medium for the FMNH

:cinnamate

reductase activity contained, beside reduced FMN, con-

siderable amounts of its oxidized form, which could in-

hibit the reaction by increasing the redox potential of

the FMNH

free

/FMN

free

pair. In contrast, virtually all FMN

was in the reduced form under steady-state conditions

in the NADH:cinnamate reductase reaction catalyzed

by d1+d3. By demonstrating full NADH:cinnamate re-

ductase activity in the presence of medium FMN, the

d1 and d3 mixture resembles yeast soluble fumarate

reductase [28]. In the latter enzyme, the roles of d1 and

d3 are played by separate enzymes, with free cytoplas-

mic flavins acting as electron carriers between them.

The modelled three-dimensional structure of

Crd. The 3D structure of heterodimeric Crd was pre-

dicted using AlphaFold2 [21]. It can be divided into

two parts connected by a flexible linker (Fig. 8). One of

them, corresponding to the d1 fragment, is formed

by CrdA and the homologous N-terminal NADH:flavin

domain of CrdB and resembles homodimeric NADH:

flavin reductases [16, 29]. The other part contains two

other CrdB domains– FAD binding2 and FMN bind.

Four flavin groups (three FMNs and one FAD in

their oxidized states) and one each of NADH and cin-

namate molecules were docked into the modeled struc-

ture using AutoDock Vina [23]. The locations of the

docked flavin groups (Fig.8) were similar to those in

the experimentally determined structures of NAD(P)H:

FMN reductase (PDBID: 1X77 [29]), the NqrC subunit

of Na

-translocating NADH:quinone oxidoreductase

(PDBID: 4XA7 [30]), and urocanate reductase (PDB ID:

6T87 [31]). These three proteins are homologous to the

NADH:flavin, FMN bind, and FAD binding2 domains of

Crd, respectively.

The d1 part of Crd contains two potential pseudo-

symmetrical NADH-binding sites near FMN

and FMN

similar to those found in the homodimeric NADH:fla-

vin reductase [16]. However, only the FMN

-adjacent

site could be occupied by NADH because the entrance

to the FMN

-adjacent site is blocked by the C-terminal

segment of CrdA (Fig. S3 in the Online Resourse 1).

Instead, the positions of the docked NADH molecule

concentrated in two areas of the protein near FMN

The shortest distance between NADH and FMN

, 10 Å,

is too long for the hydride ion transfer between them

(for comparison, the distance between NADH and

FMN

is only 4 Å). Similar docking experiments pro-

vided no evidence for a competent NADH-binding site

in the middle, FMN bind domain of CrdB. The location

of docked cinnamate in the FAD binding 2 domain of

CrdB was similar to that of urocanate in urocanate re-

ductase [31].

The edge-to-edge distance between FAD and *FMN

in the resulting model is approximately 9 Å (Fig. 8),

allowing rapid electron transfer between these fla-

vin groups, consistent with the finding that this step

is not rate-limiting in NADH:2-enoate reductases [3].

In contrast, the FMN

and *FMN groups are separated

by 24 Å in the model (Fig. 8), a distance at which rap-

id electron transfer is not feasible. Clearly, Crd should

adopt a different conformation to make the FMN

and

*FMN groups closer to each other and allow electron

transfer between them in the catalytic reaction. Inter-

estingly, the same step is rate-limiting in the electron

transfer in canonical NADH:2-enoate reductases [3],

Table 3. The catalytic activities of Crd and its deletion variants

Enzyme/variant Bound prosthetic groups

Activity, s

–1

MV:cinnamate NADH:cinnamate NADH:FMN

fCrd 2 FMN, 1 FAD, 1 *FMN

22 ± 2 15 ± 2 62 ± 15

aCrd 2 FMN, 1 FAD 0.7 ± 0.1 <0.05 58 ± 13

d1 2 FMN n.d.

<0.05 48 ± 21

d3 1 FAD 0.7 ± 0.1 <0.05 n.d.

The assay mixture contained 120µM NADH or 100 µM reduced MV as the electron donor and 50 μM cinnamate or FMN

as the electron acceptor. The activity values shown are for the reactions started by electron acceptor (the reactions started by

the enzyme demonstrated a profound time lag).

The asterisk indicates covalently bound flavin.

n.d., not determined.

BERTSOVA et al.252

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

indicating that the above distance is similarly non-op-

timal in these enzymes.

The validity of the model predicted by AlphaFold2

with the docked flavins was confirmed by MD simu-

lations (Fig. S4 in the Online Resourse 1). Specifically,

none of the four flavin groups was lost during the sim-

ulations. Furthermore, the simulations did not change

significantly the structure predicted by AlphaFold2,

except for a rigid-body movement of the d1 part, con-

nected by a flexible linker, by approximately 11 Å rel-

ative the rest of the molecule (Fig. S4, A and C in the

Online Resourse 1). This result showed that Crd struc-

ture is indeed flexible. However, the flavin-to-flavin

distances were not appreciably different in the sim-

ulated structure (Fig. S4B in the Online Resourse 1)

and remained virtually unchanged in the analogous

structure with the flavins in their reduced forms (data

notshown).

DISCUSSION

Three-domain NADH:2-enoate reductases are abun-

dant among anaerobic and facultatively anaerobic

prokaryotic and eukaryotic microorganisms, which

suggests their metabolic importance. The enzymes

with NADH:fumarate reductase activity participate in

anaerobic respiration, allowing reoxidation of glycoly-

sis-produced NADH to increase ATP production during

fermentation. This role was best demonstrated in

unicellular eukaryotes of the class Kinetoplastida, in

which fermentation yields succinate as a major prod-

uct and glycosomal and mitochondrial NADH:fuma-

rate reductase inactivation blocks completely fuma-

rate respiration [32]. NADH:2-enoate reductases with

different substrate specificities may have other roles.

Thus, NADH:acrylate reductase from marine bacteri-

um V. harveyi is apparently involved in detoxication

of acrylate [4], massively formed in marine habitats

during degradation of dimethylsulfoniopropionate,

the main compatible solute in alga.

While sharing overall structural similarity with

other microbial three-domain NADH:2-enoate reduc-

tases, such as the fumarate reductases of K. pneumoni-

ae and Kinetoplastida and acrylate reductase of V. har-

veyi, the V. ruber cinnamate reductase Crd displays

significant differences. In all these enzymes, 2-enoate

is reduced in FAD binding 2 domain by the electrons

Fig. 8. The predicted three-dimensional structure of Crd with bound FAD and three FMNs in a cartoon representation.

The domains are colored as in Fig.1a. The right part shows schematically the edge-to-edge distances between the prosthetic

groups and the proposed pathways for electron and hydride ion transfer (indicated by brown and green arrows, respectively).

CINNAMATE REDUCTASE OF V. ruber 253

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

transmitted via a covalently bound FMN residue of

FMN bind domain [3]. However, the NADH-oxidizing

activity belongs to NADH:flavin domain in Crd but to

either OYE-like domain (in K. pneumoniae NADH:fuma-

rate and V. harveyi NADH:acrylate reductases) or FAD

binding6 domain (in Kinetoplastida NADH:fumarate re-

ductases).

Furthermore, only Crd contains an additional sub-

unit (CrdA) formed by a single NADH:flavin domain,

which is homologous to the analogous domain of CrdB

(51% identity, 65% similarity). Its gene (crdA) flanks

the crdB gene in one chromosome (Fig.1c), which, to-

gether with the finding that the tag-less CrdA co-puri-

fies with 6×His-tagged CrdB on a Ni-column, provides

strong evidence that Crd functions as a tight CrdA/CrdB

complex in vivo. The observed noncovalently bound

FMN:FAD stoichiometry of 2 : 1 is fully consistent with

the heterodimeric structure of Crd. Noteworthy, pro-

karyotic NADH:flavin reductases are generally encoded

by single genes but function as homodimers contain-

ing two bound FMN molecules at the dimer interface

[16, 29]. The homodimeric structure apparently pre-

vents direct fusion of the NADH:flavin reductase do-

main with other functional domains to form an ex-

tended multidomain polypeptide.

Under aerobic conditions, three-domain NADH:

2-enoate reductases catalyze O

reduction to form re-

active oxygen species (ROS). As a striking example,

K. pneumoniae NADH:fumarate reductase oxidizes NADH

to form H

under aerobic conditions, and the oxida-

tion reaction is not accelerated by fumarate, indicat-

ing that all NADH reducing equivalents are consumed

in H

production [3]. On the other hand, ROS pro-

duction may be beneficial in some cases. Thus, ROS

generation by NADH:fumarate reductases in Kineto-

plastida may aid in completing the parasitic cycle (dif-

ferentiation of procyclics into epimastigotes) in the

insect vector [33]. However, excessive ROS generation

upon transfer to aerobiosis is expected to be harmful

for most facultatively anaerobic microorganisms. For

instance, accumulation of H

in millimolar concen-

trations under the action of NADH:flavin reductase in

aerobic conditions caused cell death in the lactic acid

bacterium Lactobacillus johnsonii [27].

The presence of a pair of NADH:flavin domains in

V. ruber Crd instead of the OYE-like or FAD binding 6

domain found in other NADH:2-enoate reductases pre-

sumably solves the problem of excessive ROS produc-

tion. The NADH:flavin domains allow a unique regula-

tion mechanism that reversibly blocks Crd activity at

high O

concentrations (Fig.7). The transition between

the active and inactive states appears to be controlled

in Crd by the redox state of one or both FMN mole-

cules in the NADH:flavin domains. This conclusion is

supported by the observations that Crd could be acti-

vated by both NADH and dithionite and that the d1 but

not d3 fragment demonstrated similar time-dependent

activation of its partial enzymatic activity.

Crd regulation appears to be associated with the

heterodimeric structure of the NADH dehydrogenase

part, formed by CrdA and the homologous N-terminal

domain of CrdB (Fig. 8). This part of Crd resembles

NADH:flavin reductases [16, 29] and similarly contains

two noncovalently bound FMN groups and two poten-

tial sites of NADH oxidation. However, the competence

of the putative NADH-binding site near FMN

seems

questionable because of the unfavorable steric effect

of the C-terminal loop of CrdA. While electron trans-

fer between FMN

and *FMN is perceivable assuming

*FMN and/or NADH:flavin dimer flexibility, the long

distance between FMN

and FMN

(Fig. 8) can hardly

decrease similarly. Hence, the electron transfer between

these flavin groups should be always much slower than

enzyme turnover [34]. These structural considerations

suggest that FMN

and FMN

are good candidates

for the catalytic and regulating groups, respectively.

Thatthese flavins have distinct roles is consistent with

significant differences in the amino acid sequenc-

es of the two NADH:flavin domains of Crd, contrast-

ing the case of homodimeric and, hence, symmetrical

NADH:flavin reductases in which two FMN sites are

identical [16]. One can further speculate that Crd ac-

tivation involves the stepwise reduction of FMN

and

FMN

causing a conformational change that forms a

functional electron transfer chain linking the donor

and acceptor.

That the electron acceptors slow down Crd activa-

tion by NADH raises the possibility that the bound ac-

ceptor competes with the regulating prosthetic group,

likely FMN

, for the electrons coming from NADH.

In this case, the activation rate should inversely cor-

relate with the rate of acceptor reduction. Such a cor-

relation was indeed observed– cinnamate was reduced

faster (Table 1) and decelerated Crd activation more

compared with acrylate (Fig. 5). In this context, the

lack of Crd activation under aerobic conditions may

mean that O

can oxidize the regulating prosthetic

group directly, without using the long electron transfer

chain involved in the case of cinnamate and acrylate.

Despite the similarity of the 2-enoate-binding ami-

no acid residues in the FAD binding 2 domains of ac-

rylate reductase and Crd (Fig. 1b), the latter enzyme

exhibits a 1000-6000-fold lower catalytic efficiency

against acrylate than against cinnamate and its deriva-

tives (Table1). Furthermore, (hydroxy)cinnamic acids,

but not acrylate, induced Crd synthesis in V. ruber

cells (Fig. 4). Clearly, the positions of amino acid res-

idues interacting with electron-acceptor carboxylates

in fumarate reductases are not the only determinants

of the substrate specificity in other NADH:2-enoate re-

ductases. The reduction of cinnamate and its deriva-

tives, toxic to cells [35], is thus the most likely physio-

BERTSOVA et al.254

BIOCHEMISTRY (Moscow) Vol. 89 No. 2 2024

logical function of Crd, and this inference is supported

by their induction of Crd synthesis in both anaerobic

and aerobic conditions (Table2). Additionally, Crd may

support anaerobic respiration of V.ruber on (hydroxy)

cinnamic acids as described for caffeyl-CoA reductase

from acetogenic bacterium Acetobacterium woodii [36].

The most effective substrates of Crd, caffeate and feru-

late, are the precursors of lignin and various pheno-

lic secondary metabolites in plants. That is why these

compounds are abundant in terrestrial niches, soil,

and animal gut in particular, but are relatively rare

in the marine habitats, where they are synthesized in

some algae [37, 38]. An interesting exception to this

rule is the seagrass meadows, which accumulate caffe-

ic acid and other phenolic compounds in rhizosphere

[35], a likely main habitat for Crd-containing marine

bacteria [39].

Crd-like reductases are relatively rare among ma-

rine microorganisms, seemingly, because (hydroxyl)

cinnamic acids are underrepresented in the habitat.

BLAST search has revealed Crd-like proteins only in

the following marine bacteria: Vibrio rhizosphaerae,

Vibrio gazogenes, Vibrio salinus, Vibrio spartinae, and

Vibrio tritonius. However, such reductases are abun-

dant among anaerobic and facultatively anaerobic

terrestrial bacteria (the Clostridium, Klebsiella, Citro-

bacter, Aeromonas, Paenibacillus, Streptococcus, and

other genera), including those forming human intesti-

nal microbiome. The presence of substantial amounts

of phenylpropionate and p-hydroxyphenylpropionate

in the mammalian intestine has long been known [40]

but their production was attributed to a different en-

zyme, bacterial two-domain 2-enoate reductase [1].

The wide distribution of Crd-like enzymes among in-

testinal bacteria suggests that they are also involved

in phenylpropionate and p-hydroxyphenylpropionate

formation in the mammalian intestine.

Two-domain 2-enoate reductases are industrial

biocatalysts increasingly used to produce chiral inter-

mediates, pharmaceuticals and agrochemicals [11, 41].

A disadvantage of these enzymes is that they are read-

ily and irreversibly inactivated by oxygen [1, 11]. The

lower sensitivity of Crd to oxygen makes it a promising

alternative for industrial application.

Contributions. AVB the conception of the study;

YVB, MVS, VAA, AAB, and AVB the acquisition and

analysis of the data; AAB and AVB writing of the man-

uscript.

Acknowledgments. MALDI MS and laser scanner

facilities became available to us in the framework of

the Moscow State University Development Program

PNG5.13. This article is devoted to cherished memory

of VladimirP. Skulachev.

Funding. This work was supported by the Russian

Science Foundation (project no.22-24-00133).

Ethics declarations. The authors declare no con-

flicts of interest. This article does not contain descrip-

tion of studies with the involvement of humans or an-

imal subjects.

Electronic supplementary material. The online

version contains supplementary material available at

https://doi.org/10.1134/S0006297924020056.

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