Role of α-Methylacyl Coenzyme A Racemase in the Degradation of Methyl-Branched Alkanes by Mycobacterium sp. Strain P101

Abstract

ABSTRACT A new isolate, Mycobacterium sp. strain P101, is capable of growth on methyl-branched alkanes (pristane, phytane, and squalane). Among ca. 10,000 Tn 5 -derived mutants, we characterized 2 mutants defective in growth on pristane or n -hexadecane. A single copy of Tn 5 was found to be inserted into the coding region of mcr (α-methylacyl coenzyme A [α-methylacyl-CoA] racemase gene) in mutant P1 and into the coding region of mls (malate synthase gene) in mutant H1. Mutant P1 could not grow on methyl-branched alkanes. The recombinant Mcr produced in Escherichia coli was confirmed to catalyze racemization of ( R )-2-methylpentadecanoyl-CoA, with a specific activity of 0.21 μmol · min −1  · mg of protein −1 . Real-time quantitative reverse transcriptase PCR analyses indicated that mcr gene expression was enhanced by the methyl-branched alkanes pristane and squalane. Mutant P1 used ( S )-2-methylbutyric acid for growth but did not use the racemic compound, and growth on n -hexadecane was not inhibited by pristane. These results suggested that the oxidation of the methyl-branched alkanoic acid is inhibited by the ( R ) isomer, although the ( R ) isomer was not toxic during growth on n -hexadecane. Based on these results, Mcr is suggested to play a critical role in β-oxidation of methyl-branched alkanes in Mycobacterium . On the other hand, mutant H1 could not grow on n -hexadecane, but it partially retained the ability to grow on pristane. The reduced growth of mutant H1 on pristane suggests that propionyl-CoA is available for cell propagation through the 2-methyl citric acid cycle, since propionyl-CoA is produced through β-oxidation of pristane.