In search for structural targets for engineering d-amino acid transaminase: modulation of pH optimum and substrate specificity

In search for structural targets for engineering d-amino acid transaminase: modulation of pH optimum and substrate specificity Full article

Journal

Biochemical Journal
ISSN: 0264-6021 , E-ISSN: 1470-8728

Output data

Year: 2023, Volume: 480, Number: 16, Pages: 1267-1284 Pages count : 18 DOI: 10.1042/bcj20230233

Authors

Shilova Sofia A ¹ , Matuta Ilya O ¹ , Khrenova Maria G ^1,2 , Nikolaeva Alena Y ^1,3 , Klyachko Natalia L ⁴ , Minyaev Mikhail E ⁵ , Khomutov Alex R ⁶ , Boyko Konstantin M ¹ , Popov Vladimir O ^1,7 , Bezsudnova Ekaterina Yu ¹

Affiliations

1	Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, Moscow, Russia
2	Department of Chemistry, Lomonosov Moscow State University, Moscow, Russia
3	Complex of NBICS Technologies, National Research Center ‘Kurchatov Institute’, Moscow, Russia
4	Moskovskij gosudarstvennyj universitet imeni M V Lomonosova, Moscow, Russia.
5	N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia.
6	Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
7	Department of Biology, Lomonosov Moscow State University, Moscow, Russia

The development of biocatalysts requires reorganization of the enzyme's active site to facilitate the productive binding of the target substrate and improve turnover number at desired conditions. Pyridoxal-5′-phosphate (PLP) - dependent transaminases are highly efficient biocatalysts for asymmetric amination of ketones and keto acids. However, transaminases, being stereoselective enzymes, have a narrow substrate specificity due to the ordered structure of the active site and work only in neutral-alkaline media. Here, we investigated the d-amino acid transaminase from Aminobacterium colombiense, with the active site organized differently from that of the canonical d-amino acid transaminase from Bacillus sp. YM-1. Using a combination of site-directed mutagenesis, kinetic analysis, molecular modeling, and structural analysis we determined the active site residues responsible for substrate binding, substrate differentiation, thermostability of a functional dimer, and affecting the pH optimum. We demonstrated that the high specificity toward d-glutamate/α-ketoglutarate is due to the interactions of a γ-carboxylate group with K237 residue, while binding of other substrates stems from the effectiveness of their accommodation in the active site optimized for d-glutamate/α-ketoglutarate binding. Furthermore, we showed that the K237A substitution shifts the catalytic activity optimum to acidic pH. Our findings are useful for achieving target substrate specificity and demonstrate the potential for developing and optimizing transaminases for various applications.

Shilova S.A. , Matuta I.O. , Khrenova M.G. , Nikolaeva A.Y. , Klyachko N.L. , Minyaev M.E. , Khomutov A.R. , Boyko K.M. , Popov V.O. , Bezsudnova E.Y.
In search for structural targets for engineering d-amino acid transaminase: modulation of pH optimum and substrate specificity
Biochemical Journal. 2023. V.480. N16. P.1267-1284. DOI: 10.1042/bcj20230233 WOS Scopus OpenAlex

≡ Web of science:	WOS:001054466200002
≡ Scopus:	2-s2.0-85168778246
≡ OpenAlex:	W4385619999