Kinetic and Bioinformatic Characterization of d-2-Hydroxyglutarate Dehydrogenase from Pseudomonas aeruginosa PAO1.

Abstract:

:d-2-Hydroxyglutarate dehydrogenase from Pseudomonas aeruginosa PAO1 (PaD2HGDH) catalyzes the oxidation of d-2-hydroxyglutarate to 2-ketoglutarate, which is a necessary step in the serine biosynthetic pathway. The dependence of P. aeruginosa on PaD2HGDH makes the enzyme a potential therapeutic target against P. aeruginosa. In this study, recombinant His-tagged PaD2HGDH was expressed and purified to high levels from gene PA0317, which was previously annotated as an FAD-binding PCMH-type domain-containing protein. The enzyme cofactor was identified as FAD with fluorescence emission after phosphodiesterase treatment and with mass spectrometry analysis. PaD2HGDH had a kcat value of 11 s-1 and a Km value of 60 μM with d-2-hydroxyglutarate at pH 7.4 and 25 °C. The enzyme was also active with d-malate but did not react with molecular oxygen. Steady-state kinetics with d-malate and phenazine methosulfate as an electron acceptor established a mechanism that was consistent with ping-pong bi-bi steady-state kinetics at pH 7.4. A comparison of the kcat/Km values with d-2-hydroxyglutarate and d-malate suggested that the C5 carboxylate of d-2-hydroxyglutarate is important for the substrate specificity of the enzyme. Other homologues of the enzyme have been previously grouped in the VAO/PMCH family of flavoproteins. PaD2HGDH shares fully conserved residues with other α-hydroxy acid oxidizing enzymes, and these conserved residues are found in the active site of the PaD2HDGH homology model. An Enzyme Function Initiative-Enzyme Similarity Tool Sequence Similarity Network analysis suggests a functional difference between PaD2HGDH and human D2HGDH, and no relationship with VAO. A phylogenetic tree analysis of PaD2HGDH, VAO, and human D2HGDH establishes genetic diversity among these enzymes.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Quaye JA,Gadda G

doi

10.1021/acs.biochem.0c00832

subject

Has Abstract

pub_date

2020-12-29 00:00:00

pages

4833-4844

issue

51

eissn

0006-2960

issn

1520-4995

journal_volume

59

pub_type

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