Effect of His₆-tag Position on the Expression and Properties of Phenylacetone Monooxygenase from Thermobifida fusca

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P. D. Parshin^1,2, A. A. Pometun^1,2,3,a*, U. A. Martysuk⁴, S. Yu. Kleymenov^3,5, D. L. Atroshenko^1,2,3, E. V. Pometun⁶, S. S. Savin^1,3, and V. I. Tishkov^1,2,3,b*

¹Lomonosov Moscow State University, Faculty of Chemistry, 119234 Moscow, Russia

²Innovations and High Technologies MSU Ltd., 109451 Moscow, Russia

³Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia

⁴Mendeleev University of Chemical Technology of Russia, 125047 Moscow, Russia

⁵Koltzov Institute of Developmental Biology of Russian Academy of Sciences, 119334 Moscow, Russia

⁶Sechenov First Moscow State Medical University, 119991 Moscow, Russia

^* To whom correspondence should be addressed.

Received March 23, 2020; Revised March 27, 2020; Accepted March 27, 2020

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Phenylacetone monooxygenase (EC 1.14.13.92, PAMО) catalyzes oxidation of ketones with molecular oxygen and NADPH with the formation of esters. PAMО is a promising enzyme for biotechnological processes. In this work, we generated genetic constructs coding for PAMO from Thermobifida fusca, containing N- or C-terminal His₆-tags (PAMO N and PAMO C, respectively), as well as PAMO L with the His₆-tag attached to the enzyme C-terminus via a 19-a.a. spacer. All PAMO variants were expressed as catalytically active proteins in Escherichia coli BL21(DE3) cells; however, the expression level of PAMO N was 3 to 5 times higher than for the other two enzymes. The catalytic constants (k_cat) of PAMO C and PAMO L were similar to that published for PAMO L produced in a different expression system; the catalytic constant for PAMO N was slightly lower (by 15%). The values of Michaelis constants with NADPH for all PAMО variants were in agreement within the published data for PAMO L (within the experimental error); however, the K_M for benzylacetone was several times higher. Thermal inactivation studies and differential scanning calorimetry demonstrated that the thermal stability of PAMO N was 3 to 4 times higher compared to that of the enzymes with the C-terminal His₆-tag.

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KEY WORDS: phenylacetone monooxygenase, His-tag, gene expression, mutagenesis, catalytic properties, temperature stability

DOI: 10.1134/S0006297920050065

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