Identification ofEscherichia coliYgaF as anl-2-Hydroxyglutarate Oxidase

Abstract

YgaF, a protein of previously unknown function in Escherichia coli, was shown to possess noncovalently bound flavin adenine dinucleotide and to exhibit L-2-hydroxyglutarate oxidase activity. The inability of anaerobic, reduced enzyme to reverse the reaction by reducing the product ␣-ketoglutaric acid is explained by the very high reduction potential (؉19 mV) of the bound cofactor. The likely role of this enzyme in the cell is to recover ␣-ketoglutarate mistakenly reduced by other enzymes or formed during growth on propionate. On the basis of the identified function, we propose that this gene be renamed lhgO.The ygaF gene of Escherichia coli is located immediately downstream of csiD, which encodes a crystallographically characterized protein of unknown function (4), and just upstream of the gabDTP operon and csiR, which encode succinic semialdehyde dehydrogenase, ␥-aminobutyric acid (GABA) transaminase, a GABA-specific permease, and a repressor (Fig. 1). The first five genes, and perhaps all six (30), are coregulated by CsiR repression and cyclic AMP-cyclic AMP receptor protein and s induction acting at csiD p during carbon starvation and at stationary phase (14,18). Expression of gabDTP is additionally controlled by s binding to gabD p1 , which is triggered by multiple-stress induction (18), and by Nac/ 70 interaction with gabD p2 in response to nitrogen starvation (30). YgaF is not obviously involved in GABA metabolism (30), and its role is unknown.On the basis of its amino acid sequence, YgaF is likely to be a flavoenzyme. It has been estimated that 1 to 3% of the identified proteins in prokaryotic and eukaryotic cells contain flavin (5) and these abundant enzymes catalyze a wide range of reactions with a diverse set of substrates, including alcohols, aldehydes, ketones, amines, dithiols, amino acids, and hydroxy acids (34). Most of these enzymes transition between the fully oxidized and two-electron reduced forms of their cofactor, but in some cases, the one-electron reduced semiquinone species is stabilized. Reoxidation of the reduced flavin coenzyme can take place via several processes, including the reaction with oxygen, as in the case of flavin oxidases. The flavin cofactors (generally, flavin mononucleotide [FMN] or flavin adenine dinucleotide [FAD]) often are tightly bound to these enzymes, and in selected examples, the coenzyme is covalently attached to the protein (11). Sequence comparisons of YgaF reveal this 422-amino-acid E. coli protein to be homologous to human mitochondrial L-2-hydroxyglutarate dehydrogenase (41% identity over 398 residues) (28), Helicobacter pylori malate:quinone oxidoreductase (24% identity over 421 residues) (33), Bacillus sp. strain B-0618 creatinase and sarcosine oxidase (23% identity over 255 residues) (32), human mitochondrial dimethylglycine dehydrogenase (24% identity over 227 residues) (2), Bacillus subtilis glycine oxidase (25% identity over 146 residues) (9), human peroxisomal L-pipecolic acid oxidase (21% identity over 219 residues) (6), and many other flavoenzymes...

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