Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 12, pp. 1775-1788 © Pleiades Publishing, Ltd., 2025.

Russian Text © The Author(s), 2025, published in Biokhimiya, 2025, Vol. 90, No. 12, pp. 1902-1916.

1775

REVIEW

Microbial 2-Enoate Reductases Containing

Covalently Bound Flavin Mononucleotide

Alexander V. Bogachev

1,a

*, Alexander A. Baykov

, Victor A. Anashkin

and Yulia V. Bertsova

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

e-mail: bogachev@belozersky.msu.ru

Received June 20, 2025

Revised August 10, 2025

Accepted August 11, 2025

Abstract— Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are prosthetic groups of many

enzymes and can be attached to proteins either covalently or non-covalently. Covalent attachment of FMN to

Thr or Ser residues via a phosphate group is catalyzed by the recently discovered enzyme flavin transferase.

Among the enzymes containing phosphoester-linked FMN, the most widely represented ones are various mi-

crobial 2-enoate reductases catalyzing reduction of unsaturated carboxylic acids (fumaric, acrylic, cinnamic,

urocanic, etc.). The review is focused on microbial 2-enoate reductases and discusses their classification by

domain organization and intracellular location, structural basis of substrate specificity, catalytic mechanism,

and function, as well as the significance and evolutionary origin of the covalent attachment of FMN as a

prosthetic group.

DOI: 10.1134/S0006297925601819

Keywords: anaerobic respiration, detoxification of 2-enoates, covalently bound FMN, catalytic mechanism

* To whom correspondence should be addressed.

INTRODUCTION

Flavin adenine dinucleotide (FAD) and flavin

mononucleotide (FMN) are the most typical pros-

thetic groups of proteins. Flavoproteins make up to

3.5% of the cellular proteome [1] and usually cata-

lyze various redox reactions [2]. Most often, flavins

are bound noncovalently, but in ~10% flavoproteins,

they are linked to the protein by a covalent bond.

In most cases, this bond is formed between the C8α

or C6 atoms of the flavin isoalloxazine ring and His,

Tyr, or Cys residues of the protein [3]. It is believed

that the attachment through the isoalloxazine ring

is autocatalytic but can be accelerated by a specific

chaperone [4, 5].

FMN can also be attached to Thr and Ser resi-

dues via a phosphoester bond with the formation of

a phosphodiester (Fig. 1) [6]. As has been shown for

the first time for the Na

-translocating NADH:quinone

oxidoreductase of the bacterial respiratory chain,

such posttranslational modification is performed by

a special enzyme, flavin transferase ApbE, which uses

FAD as a substrate [7]. ApbE recognizes the consen-

sus DgxtsAT/S motif (modified Thr or Ser residues

are shown in bold) in the target protein [7-10].

The gene for flavin transferase has been iden-

tified only in some eukaryotic genomes; it is mod-

erately common among archaea and widespread

among bacteria [7] (is present in a half of bacterial

genomes [11]). Interestingly, ApbE is found in pro-

karyotes much more frequently than the Na

-trans-

locating NADH:quinone oxidoreductase, which is an

indirect evidence of flavinylation by ApbE of other

bacterial proteins [7, 12].

In general, proteins flavinylated by ApbE have

been studied much less compared to proteins in

which flavin is bound through the isoalloxazine ring

(covalently or noncovalently). Microbial 2-enolate re-

ductases are examples of enzymes containing FMN

residues linked via a phosphoester bond. They cata-

lyze reduction of 2-enoate with the formation of an

anion of the respective saturated carboxylic acid (1):

R–CH=CH–COO

–

→

R–CH

–CH

–COO

–

. (1)

BOGACHEV et al.1776

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Fig.  1. FMN covalent attachment via a phosphate group to Ser (Thr) protein residues. C6 and C8α atoms of the isoallox-

azine ring (shown in red) can form N–C, O–C or S–C bonds with His, Tyr, or Cys residues, respectively (not discussed

in the review).

2-Enoates (anions of fumaric, acrylic, cinnamic,

urocanic, and other acids) are widely represented in

cells; the specificity of 2-enoate reductases is deter-

mined by the nature of the R substituent. In this re-

view, we discuss classification of 2-enoate reductases

according to the domain organization and intracel-

lular location, the structural bases of their substrate

specificity, the catalytic mechanism, and function of

these enzymes, as well as the significance and evolu-

tionary origin of the covalent attachment of FMN as

a prosthetic group.

DOMAIN STRUCTURE

OF MICROBIAL 2-ENOATE REDUCTASES

In prokaryotic genomes, the genes of functionally

related proteins are often grouped together [13, 14],

so that the functional relationship of these proteins

can be predicted by the analysis of the genomic con-

text [15]. In particular, genomic analysis has shown

that the apbE gene and genes of proteins containing

the FAD_binding_2 domain (Pfam ID: PF00890) are

frequently located close to each other [11]. Moreover,

these proteins usually contain either the FMN_bind

domain (PF04205[16, 17]) flavinylated by ApbE or an

amino acid sequence that includes the potential flavi-

nylation motif DgxtsAT/S (although not recognized as

an individual domain by bioinformatics algorithms)

(Fig. 2).

The FAD_binding_2 domain is widespread among

eukaryotes and prokaryotes; it is a component of suc-

cinate dehydrogenase (complex II of the respiratory

chain), fumarate reductase, aspartate oxidase, gluta-

thione reductase, and some other enzymes [18, 19].

This domain contains FAD (most often, noncovalent-

ly bound) and catalytic sites for substrate oxidation/

reduction. In most cases, the FAD_binding_2 domain

Fig.  2. a-e) Domain architecture of microbial 2-enoate re-

ductases containing covalently bound FMN (according to

the InterPro database [63]). The FAD_binding_ 2 (PF00890),

FMN_bind (PF04205), OYE-like (PF00724), FAD_binding_6

(PF00970), NADH:flavin (PF03358) and ApbE (PF02424) do-

mains are indicated by different colors; redox-active pros-

thetic groups of the domains are given within rectangles;

signal peptides are shown as small dark blue rectangles;

flavinylation motif DgxtsAT/S is shown in red (see the text

for the examples of experimentally studied proteins with

the indicated types of domain architecture).

MICROBIAL 2-ENOATE REDUCTASES 1777

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

reduces 2-enoate with the formation of respective

carboxylic acid or performs the reverse reaction,

i.e., oxidation of carboxylic acid with the formation

of respective 2-enoate. The most typical substrate of

2-enoate reductases is fumarate. It is typically reduced

by the membrane-bound quinol:fumarate oxidore-

ductase complex, which contains the FAD_binding_ 2

domain and is structurally similar to complex  II of

the respiratory chain  [20]. Only some microorganisms

use periplasmic flavocytochromes c consisting of the

FAD_binding_2 domain and the heme C-containing

cytochrome domain for anaerobic respiration; these

domains may be parts of a single polypeptide or be-

long to two different subunits. The donor of reducing

equivalents for flavocytochromes is usually low-po-

tential cytochrome c [18, 19, 21].

The functioning of the FAD_binding_2 domain is

provided by the electron transfer from an external

donor/acceptor (e.g., quinol or cytochrome  c) to FAD.

This electron transport pathway is usually formed

by hemes or FeS clusters of accessory domains or

subunits. In 2-enoate reductases shown in Fig.  2,

the transfer of electrons from the external donor

of reducing equivalents to the substrate most proba-

bly involves covalently bound FMN.

PERIPLASMIC (EXTRACELLULAR) FUMARATE

REDUCTASES AND UROCANATE REDUCTASES

OF THE ANAEROBIC RESPIRATORY CHAIN

The proteins shown in Fig.  2, a and b, con-

tain the C-terminal domain FAD_binding_2 and the

N-terminal fragment or domain capable of attaching

FMN through a phosphoester bond. The N-terminal

sequences of these proteins also contain the Sec-

or Tat-type signal peptides, suggesting periplasmic

(extracellular) location of mature proteins. In most

cases, these signal peptides include the site for the

covalent attachment of lipids. Therefore, the corre-

sponding proteins are lipoproteins linked to the outer

surface of bacterial cytoplasmic membrane.

The proteins shown in Fig.  2a are common

among anaerobic Gram-positive bacteria. In most cas-

es, these proteins are extracellular fumarate reduc-

tases of the anaerobic respiratory chain, which are

structurally similar to flavocytochromes c and differ

from them only in that the electron transfer to the

catalytic site in these enzymes involves covalently

bound FMN instead of hemes  C. This has been ver-

ified for the extracellular fumarate reductase FrdA

(UniProt ID: Q8YA11) from the pathogenic bacterium

Listeria monocytogenes[22]. Indeed, the strain of this

bacterium with the disrupted frdA gene lost the abil-

ity for anaerobic growth with fumarate as the ter-

minal electron acceptor. The functioning of FrdA re-

quires its flavinylation and the presence of a system

for the extracellular electron transfer, which seems to

be the source of reducing equivalents for fumarate

reduction in L.  monocytogenes [22].

The substrate specificity of proteins contain-

ing the FMN_bind domain in the N-terminal region

(Fig. 2b) is different. For example, the periplasmic

(extracellular) urocanate reductase UrdA (Q8CVD0)

from the bacterium Shewanella oneidensis MR-1 re-

duces urocanic acid at a high rate but is inactive to-

ward fumaric or any other natural α,β-unsaturated

carboxylic acid [23]. The functioning of this protein

allows S.  oneidensis to use urocanate as the termi-

nal electron acceptor for anaerobic respiration [23].

A natural donor of reducing equivalents for this en-

zyme is membrane-bound noncoupled menaquinol:-

cytochromec oxidoreductase CymA[24]. Similar data

were obtained for UrdA from Enterococcus rivo-

rum [22]. Flavinylation of UrdA is necessary for its

enzymatic activity and physiological function[22,23].

The structures of full-length FrdA and UrdA, not

determined experimentally, have been predicted with

AlphaFold2 [25] or Chai-1 [26] (Fig.  3, a and b). Ac-

cording to the obtained model, the N-terminal region

of FrdA (shown in black in Fig.  3a) followed by the

FAD_binding_2 domain is bound to the latter via a

flexible linker and forms an α-helix adjacent to the

main part of the enzyme. Notably, the covalently

bound FMN is close to FAD (edge-to-edge distance,

5.4Å), which should allow rapid electron transfer be-

tween the two flavins. In the model of UrdA, the co-

valently bound FMN of the FMN_bind domain is also

located in close proximity to FAD of the FAD_bind-

ing_2 domain (edge-to-edge distance, 7  Å) (Fig.  3b).

The access to the FMN residue on the surface of the

UrdA molecule is sterically hindered, which may pre-

vent its reduction by external reducers, at least large-

size ones, such as cytochrome c. Since the FAD_bind-

ing_2 and FMN_bind domains in UrdA are linked to

each other via a flexible linker (Fig.3b), the latter do-

main can presumably shift away from the FAD_bind-

ing_2 domain to accept electrons from cytochrome  c.

Therefore, we assume that the covalently bound FMN

in FrdA and UrdA acts as a mobile electron carri-

er transporting electrons from an external source of

reducing equivalents to FAD of the catalytic domain.

CYTOPLASMIC NADH:2-ENOATE

REDUCTASES OF BACTERIA

Potential flavinylated 2-enoate reductases with a

more complex domain architecture (Fig.  2, c and d)

contain the C-terminal FAD_binding_2 catalytic do-

main and the FMN_bind domain with the covalent-

ly bound FMN, which make them similar to the

BOGACHEV et al.1778

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Fig. 3. The structures of full-length 2-enoate reductases containing covalently bound FMN: a)  fumarate reductase FrdA

from L. monocytogenes; b) urocanate reductase UrdA from S.  oneidensis; c)  NADH:fumarate reductase KpFrd from Klebsiella

pneumoniae; c′)  NADH:acrylate reductase VhArd from Vibrio harveyi; d)  NADH:hydroxycinnamate reductase CrdAB from

Vibrio ruber; e)  NADH:fumarate reductase KnFrd from Leptomonas pyrrhocoris. Domains and linkers are colored as in

Fig.  2. The structures were predicted with AlphaFold2.3.2; FAD was positioned in the FAD_binding_2, ApbE, and FAD_bind-

ing_6 domains like in crystal structures (PDB codes: 1D4E, 3PND, and 2EIX, respectively); FMN was covalently bound to the

FMN_bind domain like in the 4XA7 structure, noncovalently bound to for the OYE-like domain like in the 5DXY structure,

or docked to the NADH:flavin domains with AutoDockVina  1.2.5[64]. The structure of FrdA with FMN (covalently bound to

Ser27) and FAD (panela) was obtained with Chai-1 [26]. Fumarate(FUM) and urocanate(URO) substrates were transferred

from the 1D4E and 6T87 structures; acrylate (ACR) and caffeate (CAF) were docked using AutoDock Vina. Positions of re-

dox-active cofactors and substrates are shown to the right of each model; the distances between them are given in Å; red,

FAD; orange, FMN (covalently bound FMN is designated as FMN*); green, substrate. All images are shown to the same scale.

above-described urocanate reductases of anaerobic

respiratory chain. However, they lack the signal pep-

tides and, therefore, have to be located in the cyto-

plasm. In addition, they include either OYE (old yel-

low enzyme)-like (PF00724) or NADH:flavin(PF03358)

accessory N-terminal domains that typically contain

a noncovalently bound FMN and are able to oxidize

NAD(P)H. Hence, proteins shown in Fig.2,candd are

putative cytoplasmic NAD(P)H:2-enoate reductases.

The OYE-like domain was found in the NADH:fu-

marate reductase KpFrd (B5XRB0) from the enteric

bacterium Klebsiella pneumoniae [27,  28] and in the

cytoplasmic NADH:acrylate reductase VhArd(P0DW92)

from the marine bacterium Vibrio harveyi[29] (Fig.2c).

The molecule of KpFrd contains three flavins (nonco-

valently bound FAD and FMN and covalently bound

FMN) as prosthetic groups [27]. This protein reduc-

es fumarate at high rates (~500 turnovers per sec)

when using the artificial electron donor methyl vi-

ologen. Among natural donors of reducing equiva-

lents, KpFrd oxidizes only NADH, which allows it to

catalyze the NADH:fumarate reductase reaction at a

rate of ~10 turnovers per sec under anaerobic con-

ditions [28].

The model of 3D structure of KpFrd obtained with

AlphaFold2 is shown in Fig.  3c. The distances between

the flavins in this protein are too large to maintain

a physiologically significant rate of electron transfer.

MICROBIAL 2-ENOATE REDUCTASES 1779

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Therefore, during the catalytic cycle of KpFrd, its do-

mains likely undergo conformational rearrangements,

which consequentially reduces the distances between

different pairs of the flavin prosthetic groups. The

FMN_bind domain is bound to the FAD_binding_ 2

and OYE-like domains via flexible linkers, thus al-

lowing the covalently bound FMN to move between

FAD of the FAD_binding_ 2 domain and FMN of the

OYE-like domain. Hence, the covalently bound FMN

functions as a mobile carrier of electrons between

different domains of KpFrd.

Measuring the kinetics of KpFrd reduction in

the presence of NADH has made it possible to elu-

cidate the mechanism of electron transfer in this

protein [28]. NADH oxidation in the OYE-like domain

results in the reduction of noncovalently bound FMN.

Next, the electrons are transferred to the covalent-

ly bound FMN in the FMN_bind domain and then to

noncovalently bound FAD and fumarate. The slowest

step, which limits the rate of enzyme turnover, is the

transfer of electrons between noncovalently and co-

valently bound FMN entities.

The VhArd protein encoded in the genome of

V. har veyi has identical domain architecture. It con-

tains the same prosthetic groups and is able to oxi-

dize NADH [29]. However, the spatial location of its

domains in the predicted 3D structure is different

from that in KpFrd. In VhArd, the FAD_binding_2 and

FMN_bind domains are closer to each other (Fig.  3c′);

hence, the distance between FAD and covalently

bound FMN is reduced to 7.7  Å, while the distance

between two FMN molecules is increased to 40  Å.

This observation confirms the above assumption that

the covalently bound FMN in the FMN_bind domain

can act as a mobile carrier of electrons between dif-

ferent protein domains. The structures of KpFrd and

VhArd might represent the two implemented variants

of mutual location of the domains.

The screening of natural α,β-unsaturated car-

boxylic acids has shown that VhArd readily reduc-

es acrylic and methacrylic acids, but not fumaric

and other unsaturated carboxylic acids. The catalyt-

ic efficiency of VhArd toward acrylate proved to be

330 times higher than toward methacrylate; hence,

this enzyme could be identified as NADH:acrylate

reductase [29].

Cytoplasmic NADH:(hydroxy)cinnamate reduc-

tase CrdAB (A0A1R4LHH9 and A0A1R4LHW6) from

the marine bacterium Vibrio ruber is a characterized

representative of flavoproteins with the structure

shown in Fig.  2d. This protein contains four flavin

cofactors (noncovalently bound FAD and two FMNs

and covalently bound FMN) as prosthetic groups. The

substrate specificity of CrdAB is unique, as it reduc-

es only (hydroxy)cinnamic acids: (cinnamic, ferulic,

p-coumaric, and caffeic) among natural 2-enoates[30].

Although CrdAB is structurally similar to the en-

zymes described above, the domain responsible for

NADH oxidation in this protein is the N-terminal

NADH:flavin domain.

An interesting property of Crd is that the ge-

nome of V.  r uber contains the crdA gene (Fig.  2d),

encoding a protein homologous to the NADH:flavin

domain of CrdB (51% identity), upstream of the crdB

gene. Prokaryotic NADH:flavin reductases are typical-

ly encoded by a single gene; however, they function

as homodimers that noncovalently bind two FMN

molecules (prosthetic groups) in the area of contact

between the monomers [31,  32]. The homodimeric

structure of NADH:flavin reductase seems to hinder

the fusion of this protein with other proteins with the

formation of an extended multidomain polypeptide.

It is possible that the original NADH:flavin reductase

gene had undergone duplication prior to the domain

fusion during the evolution of CrdB. Next, one of the

two resultant genes fused with the precursor of the

crdB gene, while the second one remained a separate

gene encoding an additional Crd subunit (CrdA).

Modeling the 3D structure of CrdAB [30] showed

that this protein consists of two parts connected

with a flexible linker (Fig. 3d). One part includes the

FAD_binding_2 and FMN_bind domains and is struc-

turally similar to UrdA (Fig.  3b). The second part con-

sists of the CrdA subunit and homologous N-terminal

NADH:flavin domain of the CrdB subunit. Such struc-

ture is similar to the 3D structures of homodimeric

NADH:flavin reductases [31,  32]. The edge-to-edge dis-

tance between FAD and covalently bound FMN is ~10  Å

(Fig.  3d), which should allow rapid electron transfer

between these flavin groups. Contrary to the above,

the distance between the covalently bound FMN and

the closest noncovalently bound FMN is ~22  Å (Fig.  3),

which should prevent electron transfer between them

at a rate sufficient for the catalysis of the NADH:cin-

namate reductase reaction. Therefore, to ensure elec-

tron transfer between noncovalently and covalent-

ly bound FMN molecules during its catalytic cycle,

Crd must adopt an alternative conformation that

brings these two flavin groups closer to each other.

NADH:FUMARATE REDUCTASES

OF KINETOPLASTIDS

In eukaryotic genomes, the apbE gene of flavin

transferase has been found only in some protists,

first of all, kinetoplastids. These parasitic microor-

ganisms contain glycosomal and mitochondrial forms

of NADH:fumarate reductase (KnFrd) functioning in

the matrix of the respective organelles and allow-

ing anaerobic respiration in the gut of a carrier in-

sect [33-35]. KnFrd is important for the development

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BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

of the parasite procyclic form; therefore, it is con-

sidered as one of the potential targets for treating

(preventing) various diseases caused by Trypanosoma

and Leishmania species [36, 37]. The molecule of

KnFrd contains the FAD_binding_ 2 domain for fu-

marate reduction and the FAD_binding_6 (PF00970)

domain for NADH oxidation. KnFrd also includes an

additional N-terminal domain homologous to the pro-

karyotic flavin transferase ApbE (Fig. 2e) [35]. KnFrd

produced in Saccharomyces cerevisiae [38] or Try-

panosoma brucei [39] cells contained at its N-termi-

nus covalently bound FMN attached to a conserved

Ser residue. Usually, flavinylation of proteins by FMN

is catalyzed by ApbE protein. However, in the case

of KnFrd, flavinylation occurs due to the activity of

the intrinsic ApbE domain. The ApbE domain can co-

valently attach FMN residue both to the same mole-

cule (cis-flavinylation) and to other KnFrd molecules

(trans-flavinylation), cis-flavinylation being more ef-

ficient than trans-flavinylation [39].

The model of the KnFrd 3D structure is shown in

Fig.  3e. In this enzyme, the distance (40  Å) between

the NADH dehydrogenase site (FAD in the FAD_bind-

ing_6 domain) and fumarate reductase site (FAD in

the FAD_binding_2 domain) is too long (40  Å) for di-

rect electron transfer between them. The substitution

of flavinylated Ser residue in the KnFrd sequence de-

prives the mutant protein of covalently bound flavin

and, as a consequence, leads to the complete loss of

the NADH:fumarate reductase activity[38]. Apparent-

ly, the functional role of FMN in this protein is the

transfer of reducing equivalents from the NADH de-

hydrogenase site (FAD_binding_6 domain) to its fuma-

rate reductase site (FAD_binding_2 domain). Despite

the fact that the site of FMN covalent attachment in

KnFrd has been established, the site of noncovalent

binding of the isoalloxazine ring of this prosthetic

group remains unknown. One can note a very short

length of KnFrd regions that cannot be assigned to the

conserved ApbE, FAD_binding_2, and FAD_binding_6

domains, as well as the low level of conservation of

amino acid residues in these regions [38]. Hence, the

primary structure of Frd has no regions capable of

forming one more flavin-binding domain. This is in

agreement with the idea that the covalently bound

FMN located at the flexible N-terminal linker can be

alternately found close to the FAD molecules in the

FAD_binding_6 and FAD_binding_2 domains, acting as

a mobile electron carrier between these domains.

THE ROLE OF COVALENTLY BOUND FMN

IN 2-ENOATE REDUCTASES

In 2-enoate reductases of the anaerobic respira-

tory chain (Fig.  2, a and b), covalently bound FMN

forms the pathway for the electron transport from

the external donor of reducing equivalents to the cat-

alytic 2-enoate reductase site and is a functional ana-

log of C-type hemes in flavocytochromes  c[18,  19,  21].

Iron contained in the heme is the most widespread

transition metal in the Earth’s crust. Hence, it is not

surprising that living organisms use Fe very exten-

sively for the catalysis of redox and other reactions.

For example, iron is a component of universal pros-

thetic groups such as hemes, FeS clusters, nonheme

Fe centers, etc. However, the saturation of the Earth’s

atmosphere with oxygen due to the appearance and

development of oxygenic photosynthesis has caused

large-scale oxidation of Fe

to Fe

. The low solubil-

ity of Fe(OH)

at neutral and especially alkaline pH

values has led to a significant decrease in the content

of soluble iron in many ecological niches, i.e., to the

decrease in the availability of iron for modern living

organisms.

Living organisms use several approaches to over-

come the shortage in soluble forms of iron. One of

them is replacement of iron-containing enzymes by

enzymes that lack iron but perform the same func-

tion. The classical example of such strategy is the

substitution of the [Fe-S]-containing ferredoxin for

flavin-containing flavodoxin in many bacteria and

algae, when their growth is limited by a source of

iron [40-42]. Replacement of hemes  C in flavocyto-

chromes c with covalently bound FMN might be an-

other example [11].

Why was a covalently linked flavin chosen for

this substitution? Most frequently, flavins are cova-

lently attached to proteins through the formation of a

chemical bond between the isoalloxazine ring of this

prosthetic group and an amino acid residue. It is be-

lieved that the physiological implication of such mod-

ification is an increase in the redox potential of the

flavin prosthetic group, which is necessary for the

catalytic activity of the respective flavoproteins [3].

On the contrary, FMN attachment via a phosphoester

bond occurs far from the isoalloxazine ring (Fig.  1),

so that such modification unlikely leads to signif-

icant changes in the redox properties of flavin. On

the other hand, in the overwhelming majority of

bacterial proteins, the domains that covalently bind

FMN via a phosphoester bond have a periplasmic

(extracellular) location [8, 27]. All the above leads to

the conclusion that the covalent attachment of FMN

is necessary for preventing dilution of this cofactor

by the external environment upon partial dissocia-

tion of its noncovalent complexes with periplasmic

(extracellular) proteins. The covalent attachment of

heme  C in cytochromes c has been explained in a

similar way [43], which once again emphasizes the

functional analogy between these two prosthetic

groups.

MICROBIAL 2-ENOATE REDUCTASES 1781

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Fig.  4. Active sites of 2-enoate reductases with bound substrates. Structures a and b were obtained by X-ray crys-

tallographic analysis; all other structures were predicted with AlphaFold (Fig. 3). a) Fumarate reductase Fcc

(PDB ID:

1D4E) with fumarate (FUM) [18]; b) urocanate reductase UrdA (PDB ID: 6T87) with urocanate (URO) [46]; c) hydroxy-

cinnamate reductase CrdAB with caffeate (CAF); d and e) acrylate reductases VhArd and SwArd, respectively, with ac-

rylate (ACR). Red, FAD; green, substrate (the numbering of the C-atoms of fumarate is indicated by green numbers);

black dashed lines, hydrogen bonds; red dashed lines, distances (Å) between N5 atom of FAD and C3 atom of substrate

and between N atom of Arg residue and C2 atom of substrate.

However, this explanation cannot be extended to

intracellular NADH:2-enoate reductases (Fig. 2, c-e).

Intheir cases, covalent binding of FMN via the phos-

phoester bond may result from the evolutionary

origin of NADH:2-enoate reductases from extracel-

lular reductases of the anaerobic respiratory chain

(Fig. 2b). An alternative explanation is the following.

FMN covalently linked via the phosphoester bond is

similar to biotin and lipoic acid in that the function-

al part of all these prosthetic groups is attached to

proteins via a long and flexible linker. Since biotin

and lipoate act as mobile carriers of chemical groups

during catalysis[44,45], the covalent binding of FMN

via a long and flexible linker may also contribute

to its function as a mobile carrier (in this case, of

reducing equivalents) between different domains of

NADH:2-enoate reductases.

THE CATALYTIC MECHANISM

AND SUBSTRATE SPECIFICITY

OF 2-ENOATE REDUCTASES

Understanding the mechanisms of enzymatic ca-

talysis is based primarily on the knowledge of spatial

structures of the enzymes’ active sites. For 2-enoate

reductases, this information includes the structures

of the catalytic fragments of flavocytochromec(Fcc

)

(PDB IDs: 1D4E; 1Y0P) [18,  19] and UrdA (PDB ID:

6T87) [46] from bacteria of the Shewanella genus de-

termined by X-ray analysis, predicted structures of

the full-length enzymes (Fig.  3), and results of dock-

ing of prosthetic groups and substrates in the latter

(Figs.  3 and 4). The validity of the structural predic-

tion with the AlphaFold algorithm is supported by

experimentally obtained structures of the enzymes.

BOGACHEV et al.1782

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

In the crystal structure of Fcc

 [18], fumarate is

located in the catalytic site in a close proximity to

FAD (Fig.  4a). Five amino acid residues interact with

the carboxylic groups of fumarate: His364 and Thr376

with the C4 group; Arg544, Arg401, and His503 with

the C1 group [18, 19]. The position of His364 is strict-

ly conserved in all described fumarate reductases, in-

cluding FrdA, KpFrd, and KnFrd (Fig. S1, Online Re-

source  1), so that the presence of His residue at this

position makes it possible to predict the fumarate re-

ductase activity in yet unstudied 2-enoate reductases.

The small distance (3.9  Å) between FAD and fu-

marate allows the transfer of hydride ion from the

N5 atom of flavin to the C3 atom of the substrate.

This transfer is accompanied by the proton transfer

through the pathway formed by Arg380, Glu377, and

Arg401 to the C2 atom of fumarate with the forma-

tion of succinate [18, 19]. Therefore, Arg401 not only

participates in the binding of the C1 carboxylic group

of the substrate, but also acts as a proton donor in

the reduction of fumarate. In accordance with the

above, the substitution of Arg401 for Lys resulted in

a ~10,000-fold decrease in k

cat

without any signifi-

cant effect on K

for fumarate  [19]. The C4 carboxylic

group of the bound fumarate in the active site of Fcc

is twisted and is out-of-plane of this compound. Such

conformation, being an intermediate between the re-

laxed conformations of fumarate and succinate and,

consequently, close to the transition state, apparently

facilitates substrate reduction.

This catalytic mechanism presumably also op-

erates in 2-enoate reductases with other substrate

specificities. In urocanate reductase UrdA, urocanate

binds in a close proximity to FAD (Fig. 4b) [46]. Like

in fumarate reductases, the binding of the C1 car-

boxylic group of the substrate involves Arg and His

residues (Arg560, Arg411, and His520 in UrdA from

S.  oneidensis) conserved in all 2-enoate reductases

(Fig.  S1, Online Resource  1). In addition, urocanate

binds to the protein in the twisted conformation un-

typical of the free substrate. This is achieved due to

the out-of-plane rotation of the urocanate imidazole

ring caused by its interaction with the carboxylic

groups of Asp388 and Glu177 residues. In line with

the above, the substitution of Glu177 and especially

Asp388 for Ala significantly reduced the catalytic ac-

tivity of UrdA, but had no appreciable effect on the

Michaelis constant for urocanate  [47]. Like in fuma-

rate reductases, Arg411 in UrdA acts as a proton do-

nor in urocanate reduction, and its substitution leads

to the complete loss of enzymatic activity  [47]. The

presence of negatively charged amino acids at posi-

tions corresponding to Asp388 and Glu177 of UrdA

appears to be a characteristic feature of the primary

structures of urocanate reductases, which distinguish-

es them from other 2-enoate reductases.

The 3D structures of active sites of other de-

scribed 2-enoate reductases have not been deter-

mined experimentally, but they can be quite reliably

modeled using AlphaFold (Fig.  3) followed by docking

of the substrates to the active site (Fig.  4, c-e). The

reliability of modeling is confirmed by the forma-

tion of a bond between the C1 carboxylic group of

the substrate and conserved His and Arg residues

(Fig.  S1, Online Resource  1). In addition, the distanc-

es between the N5 atom of FAD and the C3 atom of

the substrate, as well as between the N atom of con-

served Arg residue and the C2 atom of the substrate

(marked with red dashed lines in Fig.  4) in the mod-

els are similar to those determined experimentally,

which provides rapid transfer of a hydride ion and

a proton, respectively [18, 46].

The binding of the caffeate carboxylic group in

the model of the CrdAB enzyme–substrate complex

involves conserved Arg781, Arg635, and His741 res-

idues. These interactions provide a sufficiently close

positioning of caffeate relative to the N5 atom of

FAD (3.7  Å) for the hydride ion transfer. The phenolic

group of caffeate is located in a hydrophobic cavity

formed by the Met597 and Leu607 residues and is

out-of-plane of the substrate. The hydroxyl group of

caffeate in the para-position is linked through a hy-

drogen bond to the Gln612 residue, which therefore

can be considered as a determinant of specificity of

2-enoate reductases toward hydroxycinnamic acids.

Interestingly, the meta-hydroxyl group of caffeate

does not form contacts with the protein in the model

of the CrdAB structure, which is in good agreement

with the ability of CrdAB to reduce caffeic and p-cou-

maric acids with equal efficiencies [30].

The 3D model of the VhArd catalytic site also

shows acrylate binding in a close proximity (3.8  Å)

to FAD (Fig.  4d). This binding is achieved by the con-

served 2-enoate reductase triad of Arg980, His940,

and Arg834. The last residue also seems to be a pro-

ton donor in the acrylate reduction by the hydride

ion. Acrylate binding takes place only through its car-

boxylic group; therefore, the specificity of VhArd is

achieved due to the small size of the substrate-bind-

ing pocket formed by the large hydrophobic residues

Pro665, Val666, Met796, and Leu806. However, these

residues apparently do not determine the specificity

of 2-enoate reductase for acrylate, because the size of

the substrate-binding site can be reduced in different

ways. In accordance with the above, the binding site

of cytochrome c:acrylate reductase from Shewanella

woodyi (SwArd)  [48], which is similar in specifici-

ty to VhArd, is formed by other bulky hydrophobic

residues (Fig.  4e; Fig.  S1, Online Resource  1). What

calls attention is the position of Trp542 in VhArd

(Leu89 in SwArd); the occurrence of a bulky hydro-

phobic amino acid residue at this position might be

MICROBIAL 2-ENOATE REDUCTASES 1783

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

a determinant of 2-enoate reductase specificity for

acrylate (Fig. 4, d and e; Fig. S1, Online Resource 1).

Analysis of bacterial genomes demonstrates a

variability of amino acid residues of hypothetical

2-enoate reductases at positions responsible for the

binding of fumarate, urocanate, acrylate, and hydroxy-

cinnamates in the characterized homologous proteins.

This fact suggests that the respective reductases are

able to use some other compounds as terminal elec-

tron acceptors during anaerobic growth [22, 49].

THE PHYSIOLOGICAL ROLE OF 2-ENOATE

REDUCTASES IN MICROORGANISMS

The main physiological role of the above-de-

scribed extracellular (periplasmic) 2-enoate reductases

is undoubtedly anaerobic respiration. Hence, inacti-

vation of the frdA and urdA genes makes L.  mono-

cytogenes and S.  oneidensis MR-1 cells incapable of

anaerobic growth with fumarate and urocanate as

terminal electron acceptors, respectively [22,  23].

Moreover, the synthesis of UrdA in S.  oneidensis MR-1

cells is induced only under anaerobic conditions and

only in the presence of urocanate in the growth me-

dium [23], i.e., only under conditions when anaero-

bic respiration on urocanate is a beneficial process.

Anaerobic respiration allows FrdA- and UrdA-con-

taining bacteria to re-oxidize NADH formed during

fermentation. Moreover, since the electron donor

for UrdA is cytochrome c [24], the functioning of

urocanate reductase allows for additional energy

conservation in a form of transmembrane potential

difference due to the functioning of the preceding

component of the electron transport chain (complexI).

We should mention a potential significance of

UrdA-like proteins for medicine. The products of bac-

terial anaerobic respiration can accumulate in the

human intestine in large quantities and, as they are

biologically active, affect the organism[50]. For exam-

ple, Molinaro et al. [51] have shown that urocanate

respiration is not characteristic of the microflora of

healthy people but is observed in the gut of most

patients with type  2 diabetes. Moreover, it has been

established that imidazolyl propionate (the product of

urocanate reduction by intestinal microflora) is ab-

sorbed into the blood and impairs glucose tolerance

by suppressing intracellular signal transduction from

insulin due to the inhibition of mTORC1 [52]. There-

fore, the urocanate respiration of intestinal bacteria

may be not the consequence but the direct cause of

type 2 diabetes, which potentially suggests new ap-

proaches for treating this widespread disease.

The main role of intracellular NADH:fumarate

reductases, such as KpFrd of K.  pneumoniae and

KnFrd of kinetoplastids, is also anaerobic respiration.

For example, in kinetoplastids, succinate is one of

the major fermentation products, while inactivation

of the glycosomal and mitochondrial forms of KnFrd

completely prevents fumarate respiration [35]. The

synthesis of KpFrd in K.  pneumoniae is induced only

under anaerobic conditions in the presence of fuma-

rate (malate) [28], which is also in good agreement

with the involvement of this enzyme in anaerobic

respiration. Under aerobic conditions, KpFrd reduc-

es O

both in the presence and absence of fumarate.

The product of this reaction is hydrogen peroxide

that can cause cell death. However, this activity might

not be physiological, because the synthesis of KpFrd

is repressed under aerobic conditions [28].

The physiological role of intracellular NADH:fu-

marate reductases is still not quite clear. Why is it ad-

vantageous to use soluble NADH:fumarate reductase

under certain conditions, if the same reaction can be

performed with the involvement of membrane-bound

enzymes of the respiratory chain (complex I and

quinol:fumarate oxidoreductase) [53]? When the lat-

ter enzymes are active, the transfer of electrons from

NADH to fumarate is accompanied not only by the

NADH reoxidation but also by energy conservation in

a form of transmembrane potential difference, which

must be energetically more favorable compared to

the functioning of noncoupled intracellular NADH:fu-

marate reductases. The use of KnFrd in kinetoplas-

tids can be accounted for by the inability of these

microorganisms to synthesize a low-potential quinone

(menaquinone or rhodoquinone) necessary for the

membrane-associated fumarate respiration involving

the electron transport chain [54]. However, K.  pneu-

moniae cells can synthesize menaquinone [55], and

the genome of this bacterium contains full set of

genes encoding complex  I and quinol:fumarate ox-

idoreductase. Therefore, it is possible that intracel-

lular NADH:fumarate reductases perform some func-

tions in addition to anaerobic respiration. Thus, it

has been hypothesized that the generation of reactive

oxygen species by KnFrd of T.  brucei in the absence

of fumarate and in the presence of O

is required

for the differentiation of the procyclic form of this

parasite into epimastigotes [56].

On the contrary, the main physiological role of

cytoplasmic NADH:acrylate reductase VhArd and

NADH:(hydroxy)cinnamate reductase CrdAB is not

anaerobic respiration but detoxification of acrylic

and hydroxycinnamic acids. The synthesis of VhArd

and CrdAB in bacterial cells is induced in the pres-

ence of acrylate and (hydroxy)cinnamates, respective-

ly, regardless of oxygen concentration in the growth

medium [29,  30]. Acrylate is an electrophilic com-

pound capable of reacting with many cell compo-

nents, which accounts for the toxic effect of acrylic

acid[57,58]. The major natural source of free acrylic

BOGACHEV et al.1784

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

acid is dimethylsulfoniopropionate (DMSP) [59]. This

compound is used as an osmolyte by many marine al-

gae and accumulates in these organisms at very high

concentrations. Decomposition of DMSP by microbial

DMSP lyases results in the formation of acrylate [59],

which makes acrylic acid quite typical for different

marine ecological niches. The bacterium V.  har veyi

is often found in acrylate-rich niches (e.g., coral mu-

cus) and can use this compound as the sole carbon

and energy source [60]. VhArd-like proteins are also

widespread among other representatives of marine

bacteria of the genus Vibrio and apparently used to

prevent the toxic effects of acrylic acid.

Similar function can be suggested for Crd-like

NADH:(hydroxy)cinnamate reductases that reduce

toxic hydroxy derivatives of cinnamic acid [61]. The

most efficient substrates for CrdAB (caffeate and

ferulate) are precursors in the synthesis of lignin

and other phenolic secondary metabolites in terres-

trial plants. Therefore, these compounds are wide-

spread in various terrestrial habitats (e.g., soil and

intestines of herbivores). Accordingly, Crd-like pro-

teins are widespread among terrestrial anaerobic

microorganisms, including bacteria of the human gut

microbiome. These proteins are found in numerous

representatives of the genera Clostridium, Klebsiella,

Citrobacter, Aeromonas, Paenibacillus, Streptococcus,

etc. In terrestrial bacteria, Crd-like proteins might

be responsible for detoxification of hydroxycinnam-

ic acids, but this hypothesis requires experimental

verification. At the same time, reductases similar to

CrdAB occur rarely in marine organisms. For exam-

ple, the Blast search reveals Crd-like proteins only in

marine bacteria of the Vibrio genus (V.  ruber, V.  rhi-

zosphaerae, V.  ga zogenes, V.  salinus, V. spartinae, and

V.  t ritonius). The only known marine ecological niche

enriched in hydroxycinnamic acids is the rhizosphere

of marine angiosperms. It has been shown that these

plants secrete caffeic acid to the rhizosphere, together

with sucrose and various phenolic compounds [61].

It is noteworthy that the above-mentioned marine

Crd-containing bacteria can be found only in the

rhizosphere of marine angiosperms [62], so detoxi-

fication of caffeate by Crd-like proteins presumably

allows Crd-containing marine bacteria to inhabit this

sugar-rich ecological niche.

The use of the NADH:flavin domain instead of

the OYE-like domain in Crd apparently gives this

protein multiple advantages. A characteristic feature

of NADH:2-enoate reductases is that under aerobic

conditions, they spent a significant quantity of re-

ducing equivalents not for the reduction of physio-

logical electron acceptors, but for the reduction of O

with the production of reactive oxygen species [28].

For strictly anaerobic bacteria, such a feature of

NADH:2-enoate reductases may be neutral. However,

in the case of facultative anaerobes, this activity may

lead to cell damage during transition to aerobic

conditions. Notably, it has been shown that CrdAB,

in contrast to other NADH:2-enoate reductases, is a

regulated enzyme that can be converted into inac-

tive form [30]. This inactivation is regulated by the

stationary level of some reduced prosthetic group in

CrdAB and involves the NADH:flavin domain. Such

regulation allows to switch off the enzymatic activity

of CrdAB at high O

concentrations to avoid the gen-

eration of reactive oxygen species. The inactivation

is reversible and, when O

concentration decreases,

reduced Crd is slowly converted into the active form.

In conclusion, 2-enoate reductases described in

this review are widespread among anaerobic and fac-

ultative anaerobic microorganisms and perform func-

tions such as anaerobic respiration, detoxification of

2-enoates, and cell cycle regulation by reactive oxy-

gen species.

Abbreviations

ApbE flavin transferase catalyzing covalent

attachment of FMN to flavoproteins

through a phosphoester bond

CrdAB cytoplasmic NADH:(hydroxy)cinnamate

reductase of Vibrio ruber

FAD flavin adenine dinucleotide

Fcc

periplasmic fumarate reductase of bac-

teria of the Shewanella genus

FMN flavin mononucleotide

FrdA extracellular fumarate reductase

ofListeria monocytogenes

KnFrd NADH:fumarate reductase of kineto-

plastids

KpFrd cytoplasmic NADH:fumarate reductase

of Klebsiella pneumoniae

OYE old yellow enzyme

UrdA bacterial periplasmic (extracellular)

urocanate reductase

VhArd cytoplasmic NADH:acrylate reductase

of Vibrio harveyi

Supplementary information

The online version contains supplementary material

available at https://doi.org/10.1134/S0006297925601819.

Acknowledgments

The work is dedicated to the memory of Vladimir

Petrovich Skulachev.

Contributions

A.V.B. developed the study concept; A.A.B. and A.V.B.

wrote the manuscript; V.A.A., analyzed 3D protein

structures; Yu.V.B., V.A.A., and A.A.B. prepared the fig-

ures; A.V.B., A.A.B., V.A.A., and Yu.V.B. discussed and

edited the manuscript.

MICROBIAL 2-ENOATE REDUCTASES 1785

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Funding

The work was supported by the Russian Science Foun-

dation (project no.24-24-00043).

Ethics approval and consent to participate

This work does not contain any studies involving hu-

man or animal subjects.

Conflict of interest

The authors of this work declare that they have no

conflicts of interest.

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