Functional characterization of PLP fold type IV transaminase with a mixed type of activity from Haliangium ochraceum.

Authors:
Alena Yu Nikolaeva
Alena Yu Nikolaeva
Bach Institute of Biochemistry
Vladimir I Timofeev
Vladimir I Timofeev
a National Research Centre 'Kurchatov Institute'
Moskva | Russia
Tatiana V Rakitina
Tatiana V Rakitina
National Research Center "Kurchatov Institute"
Moskva | Russia
Vladimir O Popov
Vladimir O Popov
A.N. Bach Institute of Biochemistry
Russia
Ekaterina Yu Bezsudnova
Ekaterina Yu Bezsudnova
A.N. Bach Institute of Biochemistry

Biochim Biophys Acta Proteins Proteom 2019 Jun 19;1867(6):575-585. Epub 2019 Mar 19.

Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky Ave. 33, bld. 2, 119071 Moscow, Russian Federation.

Pyridoxal-5'-phosphate (PLP)-dependent transaminases are industrially important enzymes catalyzing the stereoselective amination of ketones and keto acids. Transaminases of PLP fold type IV are characterized by (R)- or (S)-stereoselective transfer of amino groups, depending on the substrate profile of the enzyme. PLP fold type IV transaminases include branched-chain amino acid transaminases (BCATs), D-amino acid transaminases and (R)-amine:pyruvate transaminases. Recently, transaminases with a mixed type of activity were identified and characterized. Here, we report biochemical and structural characterization of a transaminase from myxobacterium Haliangium ochraceum (Hoch3033), which is active towards keto analogs of branched-chain amino acids (specific substrates for BCATs) and (R)-(+)-α-methylbenzylamine (specific substrate for (R)-amine:pyruvate transaminases). The enzyme is characterized by an alkaline pH optimum (pH 10.0-10.5) and a tolerance to high salt concentrations (up to 2 M NaCl). The structure of Hoch3033 was determined at 2.35 Å resolution. The overall fold of the enzyme was similar to those of known enzymes of PLP fold type IV. The mixed type of activity of Hoch3033 was implemented within the BCAT-like active site. However, in the active site of Hoch3033, we observed substitutions of specificity-determining residues that are important for substrate binding in canonical BCATs. We suggest that these changes result in the loss of activity towards α-ketoglutarate and increase the affinity towards (R)-(+)-α-methylbenzylamine. These results complement our knowledge of the catalytic diversity of transaminases and indicate the need for further research to understand the structural basis of substrate specificity in these enzymes.

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http://dx.doi.org/10.1016/j.bbapap.2019.03.005DOI Listing
June 2019
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