Moenomycin Biosynthesis: Structure and Mechanism of Action of the Prenyltransferase MoeN5

Abstract

We report the structure of MoeN5, a unique prenyl transferase involved in moenomycin biosynthesis. MoeN5 catalyzes the reaction of geranyl diphosphate (GPP) with the cis-farnesyl group in phosphoglycolipid 5 to form the (C 25 ) moenocinyl sidechain-containing lipid 7. GPP binds to an allylic site (S1) and aligns well with known S1 site inhibitors. Alkyl glycosides, glycolipids, can bind to both S1 as well as a second site, S2. Long sidechains in S2 are "bent" and co-locate with the homoallylic substrate isopentenyl diphosphate in other prenyl transferases. These observations support a MoeN5 mechanism in which 5 binds to S2 with its C6-C11 group poised to attack C1 in GPP to form the moenocinyl sidechain, the more distal regions of 5 aligning with the distal glucose in decyl maltoside. The results are of general interest because they provide the first structures of MoeN5 and a structural basis for its mechanism of action, results that will facilitate the design of new antibiotics. Keywords isoprenoid biosynthesis; protein structure; antibiotics; enzyme mechanisms; drug discoveryThe moenomycins are a potentially important class of antibiotics, discovered ~60 years ago [1] , that function by inhibiting bacterial cell wall biosynthesis [2] . Moenomycin A (MmA 1, Scheme 1) is one member of this class and in vitro is a more potent antibiotic than is vancomycin. It has, however, poor pharmacokinetics, but given the global increase in antibiotic resistance [3] there is renewed interest in the development of MmA analogs [4] , as well as the possibility of using MmA to treat gastro-intestinal tract infections, such as that caused by Helicobacter pylori [5] . MmA biosynthesis is quite complex, with 17 enzymes involved [4a] . The first committed step involves condensation of 3-phosphoglycerate (2) with farnesyl diphosphate (3) to form 2-(Z,E)-farnesyl-3-phosphoglycerate [FPG, 4] in a reaction catalyzed by MoeO5 [6] and in recent work we reported the X-ray structure of MoeO5 and proposed a mechanism of action [6b] . 4 then undergoes a series of glycosylations and other modifications to form a phosphoglycolipid, the FPG-trisaccharide 5 which then reacts with geranyl diphosphate (GPP, 6) to produce the (C 25 ) moenocinyl trisaccharide 7 [7] which after further transformations [4a] results in formation of 1. The 5 → 7 reaction is catalyzed by MoeN5 [4a] and has been proposed [4a] to involve two carbocations. However, the structure of MoeN5 has not been reported and a BLAST (Basic Local Alignment Search Tool) search reveals no homologs with known structure. We thus sought to determine the structure of MoeN5-in the absence and presence of GPP, FPG and a series of model glycolipids-in Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript order to help clarify the mechanism of action of this very unusual "head-to-middle" prenyl transferase.Recombinant MoeN5 from Streptomyces ghanaensis was first expressed with a 35-residue N-terminal tag, hereinafter denoted as MoeN5-NT, and its structure was so...

Structure, catalysis, and inhibition mechanism of prenyltransferase

“…MoeN5 can catalyze one GPP molecule (S1 site) and the cis ‐farnesyl group in the phosphoglycolipid (FPG‐trisaccharide; S2 site) through head‐to‐middle condensation to produce the 25‐carbon moenocinyl‐side‐chain‐containing lipid in the biosynthesis of moenomycin (Figure 7). The crystal structure and reaction mechanism of MoeN5 were recently reported by Zhang et al 20 MoeN5 forms a dimer both in the solution and in the crystal structure. Unlike the previously mentioned head‐to‐middle enzymes, MoeN5 exhibits a structural similarity to head‐to‐tail trans ‐prenyl synthases, such as geranyl transferase and GGPPS, which generally contain two canonical aspartate‐rich domains, DDXXD and DXXXD, coordinating Mg 2+ ion for the ionization of allylic substrates.…”

“…Unlike the previously mentioned head‐to‐middle enzymes, MoeN5 exhibits a structural similarity to head‐to‐tail trans ‐prenyl synthases, such as geranyl transferase and GGPPS, which generally contain two canonical aspartate‐rich domains, DDXXD and DXXXD, coordinating Mg 2+ ion for the ionization of allylic substrates. Crystal structures of several ligand‐bound complexes of MoeN5 provide a structural basis for a rational catalytic reaction mechanism, in which the long side‐chain of FPG‐trisaccharide exhibits a unique bent conformation (C6–C11) to attack the C1 of GPP, followed by the formation of intermediate of 5‐ and 6‐membered‐ring carbocations and then the 25‐carbon moenocinyl side‐chain 20 …”

“…Two FPP molecules are catalyzed through two‐step reductive head‐to‐head condensation by squalene synthase (SQS) to form squalene, a precursor in cholesterol and steroid hormone synthesis in plants and animals 17,18 . One GPP molecule and the cis ‐farnesyl group in phosphoglycolipid (FPG‐ trisaccharide) are catalyzed by a unique head‐to‐middle prenyltransferase, MoeN5, to produce the 25‐carbon moenocinyl side‐chain–containing lipid in moenomycin biosynthesis 19,20 . Terpenoid cyclase catalyzes FPP cyclization to form the cyclic sesquiterpene hydrocarbon trichodiene, a precursor in antibiotic and mycotoxin biosynthesis 21 .…”