The gene ncgl2918 encodes a novel maleylpyruvate isomerase that needs mycothiol as cofactor and links mycothiol biosynthesis and gentisate assimilation in Corynebacterium glutamicum.
Data mining of the Corynebacterium glutamicum genome identified 4 genes analogous to the mshA, mshB, mshC, and mshD genes that are involved in biosynthesis of mycothiol in Mycobacterium tuberculosis and Mycobacterium smegmatis. Individual deletion of these genes was carried out in this study. Mutants mshC- and mshD- lost the ability to produce mycothiol, but mutant mshB- produced mycothiol as the wild type did. The phenotypes of mutants mshC- and mshD- were the same as the wild type when grown in LB or BHIS media, but mutants mshC- and mshD- were not able to grow in mineral medium with gentisate or 3-hydroxybenzoate as carbon sources. C. glutamicum assimilated gentisate and 3-hydroxybenzoate via a glutathione-independent gentisate pathway. In this study it was found that the maleylpyruvate isomerase, which catalyzes the conversion of maleylpyruvate into fumarylpyruvate in the glutathione-independent gentisate pathway, needed mycothiol as a cofactor. This mycothiol-dependent maleylpyruvate isomerase gene (ncgl2918) was cloned, actively expressed, and purified from Escherichia coli. The purified mycothiol-dependent isomerase is a monomer of 34 kDa. The apparent Km and Vmax values for maleylpyruvate were determined to be 148.4 +/- 11.9 microM and 1520 +/- 57.4 micromol/min/mg, respectively (mycothiol concentration, 2.5 microM). Previous studies had shown that mycothiol played roles in detoxification of oxidative chemicals and antibiotics in streptomycetes and mycobacteria. To our knowledge, this is the first demonstration that mycothiol is essential for growth of C. glutamicum with gentisate or 3-hydroxybenzoate as carbon sources and the first characterization of a mycothiol-dependent maleylpyruvate isomerase.
Feng
Jie
J
State Key Laboratory of Microbial Resources at Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China.
Che
Yongsheng
Y
Milse
Johanna
J
Yin
Ya-Jie
YJ
Liu
Lei
L
Rückert
Christian
C
Shen
Xi-Hui
XH
Qi
Su-Wei
SW
Kalinowski
Jörn
J
Liu
Shuang-Jiang
SJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002244:Carbon / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D048230:Corynebacterium glutamicum / Q000378:metabolism*; D003545:Cysteine; D017931:DNA Primers / Q000737:chemistry; D004187:Disaccharides / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005841:Gentisates / Q000378:metabolism*; D006020:Glycopeptides; D062385:Hydroxybenzoates / Q000737:chemistry / Q000378:metabolism; D007294:Inositol; D007477:Ions; D007700:Kinetics; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D010641:Phenotype; D010867:Pimelic Acids / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011720:Pyrazoles / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D019745:cis-trans-Isomerases / Q000235:genetics* / Q000502:physiology*
D001426:Bacterial Proteins; D017931:DNA Primers; D004187:Disaccharides; D005841:Gentisates; D006020:Glycopeptides; D062385:Hydroxybenzoates; D007477:Ions; D010867:Pimelic Acids; D011720:Pyrazoles; D013438:Sulfhydryl Compounds; C113957:maleylpyruvic acid; C089265:mycothiol; C032948:3-hydroxybenzoic acid; D007294:Inositol; D002244:Carbon; D019745:cis-trans-Isomerases; C046138:maleylpyruvate isomerase; D003545:Cysteine