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Journal of Bacteriology, November 2004, p. 7214-7220, Vol. 186, No. 21
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.21.7214-7220.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa Sakyo-ku, Kyoto,¹ New Products & Technology Lab, COSMO Research Institute, Satte, Saitama, Japan,² Katholieke Universiteit Leuven, Campus Gasthuisberg, Department Moleculaire Celbiologie, Afdeling Farmakologie, Leuven, Belgium³

Received 30 June 2004/ Accepted 1 August 2004

A new isolate, Mycobacterium sp. strain P101, is capable of growth on methyl-branched alkanes (pristane, phytane, and squalane). Among ca. 10,000 Tn5-derived mutants, we characterized 2 mutants defective in growth on pristane or n-hexadecane. A single copy of Tn5 was found to be inserted into the coding region of mcr (-methylacyl coenzyme A [-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 ß-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 ß-oxidation of pristane.

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