Comparative analysis of mycobacterial NADH pyrophosphatase isoforms reveals a novel mechanism for isoniazid and ethionamide inactivation.
NADH pyrophosphatase (NudC) catalyses the hydrolysis of NAD(H) to AMP and NMN(H) [nicotinamide mononucleotide (reduced form)]. NudC multiple sequence alignment reveals that homologues from most Mycobacterium tuberculosis isolates, but not other mycobacterial species, have a polymorphism at the highly conserved residue 237. To elucidate the functional significance of this polymorphism, comparative analyses were performed using representative NudC isoforms from M. tuberculosis H37Rv (NudC(Rv)) and M. bovis BCG (NudC(BCG)). Biochemical analysis showed that the P237Q polymorphism prevents dimer formation, and results in a loss of enzymatic activity. Importantly, NudC(BCG) was found to degrade the active forms of isoniazid (INH), INH-NAD and ethionamide (ETH), ETH-NAD. Consequently, overexpression of NudC(BCG) in Mycobacterium smegmatis mc(2)155 and M. bovis BCG resulted in a high level of resistance to both INH and ETH. Further genetic studies showed that deletion of the nudC gene in M. smegmatis mc(2)155 and M. bovis BCG resulted in increased susceptibility to INH and ETH. Moreover, inactivation of NudC in both strains caused a defect in drug tolerance phenotype for both drugs in exposure assays. Taken together, these data suggest that mycobacterial NudC plays an important role in the inactivation of INH and ETH.
State Key Laboratory of Virology Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
Xu-De
XD
Wang
Jing
J
Gu
Ting
T
Wang
Li-Jun
LJ
Bi
Zhi-Ping
ZP
Zhang
Zong-Qiang
ZQ
Cui
Hong-Ping
HP
Wei
Jiao-Yu
JY
Deng
Xian-En
XE
Zhang
D000595:Amino Acid Sequence; D000995:Antitubercular Agents / Q000378:metabolism* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005000:Ethionamide / Q000378:metabolism* / Q000494:pharmacology; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007538:Isoniazid / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D009163:Mycobacterium bovis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D020102:Mycobacterium smegmatis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D007527:Isoenzymes; D011755:Pyrophosphatases; C021899:nucleotide pyrophosphatase; D005000:Ethionamide; D007538:Isoniazid