TY - JOUR
T1 - The Crystal Structure of the Novobiocin Biosynthetic Enzyme NovP
T2 - The First Representative Structure for the TylF O-Methyltransferase Superfamily
AU - Gómez García, Inmaculada
AU - Stevenson, Clare E.M.
AU - Usón, Isabel
AU - Freel Meyers, Caren L.
AU - Walsh, Christopher T.
AU - Lawson, David M.
N1 - Funding Information:
I.G.G., C.E.M.S., and D.M.L. were supported by the Biotechnology and Biological Sciences Research Council through responsive mode funding (ref. B19400) and by a Core Strategic Grant to the John Innes Center. I.U. was supported by the European Union Integrated Project BIOXHIT and the Spanish MEC (grant BIO2006-14139). C.L.F.M. and C.T.W. were supported by National Institutes of Health grants F32 AI054007 and GM 20011, respectively. We are grateful to L. Heide for critically reading this manuscript and to M. Cianci, M. Ellis, and R. Strange for assistance with data collection at the SRS.
PY - 2010/1/15
Y1 - 2010/1/15
N2 - NovP is an S-adenosyl-l-methionine-dependent O-methyltransferase that catalyzes the penultimate step in the biosynthesis of the aminocoumarin antibiotic novobiocin. Specifically, it methylates at 4-OH of the noviose moiety, and the resultant methoxy group is important for the potency of the mature antibiotic: previous crystallographic studies have shown that this group interacts directly with the target enzyme DNA gyrase, which is a validated drug target. We have determined the high-resolution crystal structure of NovP from Streptomyces spheroides as a binary complex with its desmethylated cosubstrate S-adenosyl-l-homocysteine. The structure displays a typical class I methyltransferase fold, in addition to motifs that are consistent with a divalent-metal-dependent mechanism. This is the first representative structure of a methyltransferase from the TylF superfamily, which includes a number of enzymes implicated in the biosynthesis of antibiotics and other therapeutics. The NovP structure reveals a number of distinctive structural features that, based on sequence conservation, are likely to be characteristic of the superfamily. These include a helical 'lid' region that gates access to the cosubstrate binding pocket and an active center that contains a 3-Asp putative metal binding site. A further conserved Asp likely acts as the general base that initiates the reaction by deprotonating the 4-OH group of the noviose unit. Using in silico docking, we have generated models of the enzyme-substrate complex that are consistent with the proposed mechanism. Furthermore, these models suggest that NovP is unlikely to tolerate significant modifications at the noviose moiety, but could show increasing substrate promiscuity as a function of the distance of the modification from the methylation site. These observations could inform future attempts to utilize NovP for methylating a range of glycosylated compounds.
AB - NovP is an S-adenosyl-l-methionine-dependent O-methyltransferase that catalyzes the penultimate step in the biosynthesis of the aminocoumarin antibiotic novobiocin. Specifically, it methylates at 4-OH of the noviose moiety, and the resultant methoxy group is important for the potency of the mature antibiotic: previous crystallographic studies have shown that this group interacts directly with the target enzyme DNA gyrase, which is a validated drug target. We have determined the high-resolution crystal structure of NovP from Streptomyces spheroides as a binary complex with its desmethylated cosubstrate S-adenosyl-l-homocysteine. The structure displays a typical class I methyltransferase fold, in addition to motifs that are consistent with a divalent-metal-dependent mechanism. This is the first representative structure of a methyltransferase from the TylF superfamily, which includes a number of enzymes implicated in the biosynthesis of antibiotics and other therapeutics. The NovP structure reveals a number of distinctive structural features that, based on sequence conservation, are likely to be characteristic of the superfamily. These include a helical 'lid' region that gates access to the cosubstrate binding pocket and an active center that contains a 3-Asp putative metal binding site. A further conserved Asp likely acts as the general base that initiates the reaction by deprotonating the 4-OH group of the noviose unit. Using in silico docking, we have generated models of the enzyme-substrate complex that are consistent with the proposed mechanism. Furthermore, these models suggest that NovP is unlikely to tolerate significant modifications at the noviose moiety, but could show increasing substrate promiscuity as a function of the distance of the modification from the methylation site. These observations could inform future attempts to utilize NovP for methylating a range of glycosylated compounds.
KW - O-methyltransferase
KW - Streptomyces spheroides
KW - TylF superfamily
KW - crystal structure
KW - novobiocin
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U2 - 10.1016/j.jmb.2009.10.045
DO - 10.1016/j.jmb.2009.10.045
M3 - Article
C2 - 19857499
AN - SCOPUS:73149115504
SN - 0022-2836
VL - 395
SP - 390
EP - 407
JO - Journal of molecular biology
JF - Journal of molecular biology
IS - 2
ER -