Cloning, mutagenesis, and physiological effect of a hydroxypyruvate reductase gene from Methylobacterium extorquens AM1.
The gene encoding the serine cycle hydroxypyruvate reductase of Methylobacterium extorquens AM1 was isolated by using a synthetic oligonucleotide with a sequence based on a known N-terminal amino acid sequence. The cloned gene was inactivated by insertion of a kanamycin resistance gene, and recombination of this insertion derivative with the wild-type gene produced a serine cycle hydroxypyruvate reductase null mutant. This mutant had lost its ability to grow on C-1 compounds but retained the ability to grow on C-2 compounds, showing that the hydroxypyruvate reductase operating in the serine cycle is not involved in the conversion of acetyl coenzyme A to glycine as previously proposed. A second hydroxypyruvate-reducing enzyme with a low level of activity was found in M. extorquens AM1; this enzyme was able to interconvert glyoxylate and glycollate. The gene encoding hydroxypyruvate reductase was shown to be located about 3 kb upstream of two other serine cycles genes encoding phosphoenolpyruvate carboxylase and malyl coenzyme A lyase.
Chistoserdova
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W. M. Keck Laboratories, California Institute of Technology, Pasadena 91125.
Lidstrom
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D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D012696:Glycine Hydroxymethyltransferase / Q000378:metabolism; D006038:Glyoxylates / Q000378:metabolism; D006088:Gram-Negative Aerobic Bacteria / Q000235:genetics* / Q000378:metabolism; D050537:Hydroxypyruvate Reductase; D007526:Isoelectric Point; D008293:Malates / Q000378:metabolism; D000432:Methanol / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010641:Phenotype; D010730:Phosphoenolpyruvate Carboxylase / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D012694:Serine / Q000378:metabolism; D000637:Transaminases / Q000378:metabolism
D006038:Glyoxylates; D008293:Malates; D012694:Serine; D000429:Alcohol Oxidoreductases; D050537:Hydroxypyruvate Reductase; D012696:Glycine Hydroxymethyltransferase; D000637:Transaminases; C022636:serine-glyoxylate aminotransferase; D010730:Phosphoenolpyruvate Carboxylase; D000432:Methanol