The Crystal Structure of the Novobiocin Biosynthetic Enzyme NovP: The First Representative Structure for the TylF O-Methyltransferase Superfamily

Abstract

SummaryNovP is an S-adenosyl-L-methionine-dependent O-methyltransferase that catalyses the penultimate step in the biosynthesis of the aminocoumarin antibiotic novobiocin. Specifically, it methylates at the 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 co-substrate, 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 co-substrate binding pocket, and an active centre 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 utilise NovP for methylating a range of glycosylated compounds.

Structural Basis of Substrate Specificity and Regiochemistry in the MycF/TylF Family of Sugar O-Methyltransferases.

“…SAM/SAH binding to MycF closes the helical lid domain over the co-substrate, partially orders the lid loop, and forms the binding site for the substrate mycinamicin III ( 2 , Figure 2d), which orders the remainder of the lid loop (Figure 2e). In contrast to the full assembly of the MycF active site, the NovP/SAH complex has a partially disordered lid loop and no metal ion 23 . The ordering upon SAM binding fully buries the co-substrate inside MycF, consistent with the reported ordered bi-bi mechanism of TylF, a MycF homolog where co-substrate exchange is the first and last step in the catalytic cycle 24 .…”

“…of 0.41 Å for 203 Cα atoms, 54% sequence identity) 5, 23 . Both in solution and in crystals, MycF is a dimer with an N-terminal helix (residues 6–19) at the subunit interface (Figure 2b).…”

“…MycF has an active site similar to those of other metal-dependent O -methyltransferases (OMT), including catechol OMT 31 , alfalfa caffeoyl coenzyme A 3-OMT 32 , the TDP-rhamnose 3-OMT CalS11 28 , the novobiocin 4′-OMT NovP 23 , and the mycinamicin 2′-OMT MycE 20 . These metal-dependent OMTs share a conserved SAM binding site, metal binding site and catalytic base position (Lys in catechol OMT and caffeoyl CoA OMT, His in MycE, and Asp in CalS11, NovP and MycF.)…”

“…The 13 experimentally characterized subfamily members are sugar 3′- and 4′-OMTs that act after the glycosyltransfer step in the biosynthesis of secondary metabolites. We modeled the substrate complex for the 4′-OMT NovP (2WK1) 23 , the only other subfamily member of known structure. The substrate DDN ( 5 ) was manually positioned in the NovP active site constrained by the methylation site and Mg 2+ coordination geometry of MycF (Figure 5a).…”

“…Within this subfamily, the substrate- and metal-free structure of NovP 23 and the kinetic mechanism of the homolog TylF 24, 25 have been reported, however the structural basis of substrate specificity and regioselectivity is unknown 20, 23 . Recent structures of natural product sugar methyltransferases, including TcaB9 26 , RebM 27 , CalS11 28 , MycE 20 and the MycF homolog NovP 23 , reveal a diverse array of scaffolds, as might be expected, given their highly divergent sequences. They share the Rossmann-like fold common to Class-I methyltransferases, but differ in the elaborations that form oligomer interfaces and confer substrate specificity 5 .…”