Antibiotic glycosyltransferases.

In the biosynthesis of several classes of antibiotics, sugars are attached to aglycone scaffolds by antibiotic-specific glycosyltransferases in the latter stages of the pathways. Two glycosylation pathways will be examined: the glycopeptide antibiotics of the vancomycin class and the aminocoumarin antibiotics of the novobiocin class. An oxidatively cross-linked heptapeptide scaffold is sequentially glucosylated and vancosaminylated by GtfE and GtfD, respectively, in vancomycin maturation, while in chloroeremomycin assembly the same heptapeptide is glucosylated by GtfB, then epivancosaminylated at two distinct sites by GtfA and GtfC. The specificity and mechanism of these glycosyltransferases will be discussed. In novobiocin biosynthesis, three enzymes (NovM, NovP and NovN) are thought to act sequentially to transfer an L-noviose moiety to the novobiocic acid aglycone (NovM), followed by 4'-hydroxyl methylation (NovP) and 3'-hydroxyl carbamoylation to produce the mature antibiotic structure, targeting the GyrB subunit of DNA gyrase. Initial characterization of NovM and NovP will be discussed.

Tailoring enzymes that modify nonribosomal peptides during and after chain elongation on NRPS assembly lines.

Nonribosomal peptide synthetases are large enzyme complexes that synthesize a variety of peptide natural products through a thiotemplated mechanism. Assembly of the peptides proceeds through amino acid loading, amide-bond formation and chain translocation, and finally thioester lysis to release the product. The final products are often heavily modified, however, through methylation, epimerization, hydroxylation, heterocyclization, oxidative cross-linking and attachment of sugars. These activities are the province of specialized enzymes (either embedded in the multidomain nonribosomal peptide synthetase structure or standalone).

Structure of the UDP-glucosyltransferase GtfB that modifies the heptapeptide aglycone in the biosynthesis of vancomycin group antibiotics.

BACKGROUND: Members of the vancomycin group of glycopeptide antibiotics have an oxidatively crosslinked heptapeptide scaffold decorated at the hydroxyl groups of 4-OH-Phegly4 or beta-OH-Tyr6 with mono- (residue 6) or disaccharides (residue 4). The disaccharide in vancomycin itself is L-vancosamine-1,2-glucose, and in chloroeremomycin it is L-4-epi-vancosamine-1,2-glucose. The sugars and their substituents play an important role in efficacy, particularly against vancomycin-resistant pathogenic enterococci. RESULTS: The glucosyltransferase, GtfB, that transfers the glucose residue from UDP-glucose to the 4-OH-Phegly4 residue of the vancomycin aglycone, initiating the glycosylation pathway in chloroeremomycin maturation, has been crystallized, and its structure has been determined by X-ray analysis at 1.8 A resolution. The enzyme has a two-domain structure, with a deep interdomain cleft identified as the likely site of UDP-glucose binding. A hydrophobic patch on the surface of the N-terminal domain is proposed to be the binding site of the aglycone substrate. Mutagenesis has revealed Asp332 as the best candidate for the general base in the glucosyltransfer reaction. CONCLUSIONS: The structure of GtfB places it in a growing group of glycosyltransferases, including Escherichia coli MurG and a beta-glucosyltransferase from T4 phage, which together form a subclass of the glycosyltransferase superfamily and give insights into the recognition of the NDP-sugar and aglycone cosubstrates. A single major interdomain linker between the N- and C- terminal domains suggests that reprogramming of sugar transfer or aglycone recognition in the antibiotic glycosyltransferases, including the glycopeptide and also the macrolide antibiotics, will be facilitated by this structural information.

Tandem action of glycosyltransferases in the maturation of vancomycin and teicoplanin aglycones: novel glycopeptides.

The glycopeptides vancomycin and teicoplanin are clinically important antibiotics. The carbohydrate portions of these molecules affect biological activity, and there is great interest in developing efficient strategies to make carbohydrate derivatives. To this end, genes encoding four glycosyltransferases, GtfB, C, D, E, were subcloned from Amycolatopsis orientalis strains that produce chloroeremomycin (GtfB, C) or vancomycin (GtfD, E) into Escherichia coli. After expression and purification, each glycosyltransferase (Gtf) was characterized for activity either with the aglycones (GtfB, E) or the glucosylated derivatives (GtfC, D) of vancomycin and teicoplanin. GtfB efficiently glucosylates vancomycin aglycone using UDP-glucose as the glycosyl donor to form desvancosaminyl-vancomycin (vancomycin pseudoaglycone), with k(cat) of 17 min(-1), but has very low glucosylation activity, < or = 0.3 min(-1), for an alternate substrate, teicoplanin aglycone. In contrast, GtfE is much more efficient at glucosylating both its natural substrate, vancomycin aglycone (k(cat) = 60 min(-1)), and an unnatural substrate, teicoplanin aglycone (k(cat) = 20 min(-1)). To test the addition of the 4-epi-vancosamine moiety by GtfC and GtfD, synthesis of UDP-beta-L-4-epi-vancosamine was undertaken. This NDP-sugar served as a substrate for both GtfC and GtfD in the presence of vancomycin pseudoaglycone (GtfC and GtfD) or the glucosylated teicoplanin scaffold, 7 (GtfD). The GtfC product was the 4-epi-vancosaminyl form of vancomycin. Remarkably, GtfD was able to utilize both an unnatural acceptor, 7, and an unnatural nucleotide sugar donor, UDP-4-epi-vancosamine, to synthesize a novel hybrid teicoplanin/vancomycin glycopeptide. These results establish the enzymatic activity of these four Gtfs, begin to probe substrate specificity, and illustrate how they can be utilized to make variant sugar forms of both the vancomycin and the teicoplanin class of glycopeptide antibiotics.

Deoxysugars in glycopeptide antibiotics: enzymatic synthesis of TDP-L-epivancosamine in chloroeremomycin biosynthesis.

The 2,3,6-trideoxysugar l-epivancosamine is the terminal sugar added to the aglycone scaffold in chloroeremomycin, a member of the vancomycin family of glycopeptide antibiotics. Five proteins from the chloroeremomycin biosynthetic cluster, ORF14 and ORF23 to ORF26, have been expressed heterologously in Escherichia coli and purified to near homogeneity, and each has been characterized for an enzymatic activity. These five enzymes reconstitute the complete biosynthesis of TDP-l-epivancosamine from TDP-4-keto-6-deoxy-d-glucose. This process involves C-2 deoxygenation, C-3 amination and methylation, C-5 epimerization, and C-4 ketoreduction. Intermediates and the final product of this pathway have been identified by mass spectrometry and NMR. The pathway established here represents the complete in vitro reconstitution of an unusual sugar for an important class of antibiotics and sets the groundwork for future combinatorial biosynthesis for new bioactive compounds.

The EntF and EntE adenylation domains of Escherichia coli enterobactin synthetase: sequestration and selectivity in acyl-AMP transfers to thiolation domain cosubstrates.

Enterobactin, the tris-(N-(2,3-dihydroxybenzoyl)serine) trilactone siderophore of Escherichia coli, is synthesized by a three-protein (EntE, B, F) six-module nonribosomal peptide synthetase (NRPS). In this work, the 142-kDa four-domain protein EntF was bisected into two double-domain fragments: a 108-kDa condensation and adenylation construct, EntF C-A, and a 37-kDa peptidyl carrier protein (PCP) and thioesterase protein, EntF PCP-TE. The adenylation domain activity of EntF C-A formed seryl-AMP but lost the ability to transfer the seryl moiety to the cognate EntF PCP-TE in trans. Seryl transfer to heterologous PCP protein fragments, the SrfB1 PCP from surfactin synthetase and Ybt PCP1 from yersiniabactin synthetase, was observed at rates of 0.5 min(-1) and 0.01 min(-1), respectively. The possibility that these slow acylation rates reflected dissociation of acyl/aminoacyl-AMP followed by adventitious thiolation by the heterologous PCPs in solution was addressed by measuring catalytic turnover of pyrophosphate (PP(i)) released from the adenylation domain. The holo SrfB1 PCP protein as well as Ybt PCP1 did not stimulate an increase in PP(i) release from EntF C-A or EntE. In this light, aminoacylations in trans between A and PCP domain fragments of NRPS assembly lines must be subjected to kinetic scrutiny to determine whether transfer is truly between protein domains or results from slow aminoacyl-AMP release and subsequent nonenzymatic thiol capture.

Assembly line enzymology by multimodular nonribosomal peptide synthetases: the thioesterase domain of E. coli EntF catalyzes both elongation and cyclolactonization.

BACKGROUND: EntF is a 142 kDa four domain (condensation-adenylation-peptidyl carrier protein-thioesterase) nonribosomal peptide synthetase (NRPS) enzyme that assembles the Escherichia coli N-acyl-serine trilactone siderophore enterobactin from serine, dihydroxybenzoate (DHB) and ATP with three other enzymes (EntB, EntD and EntE). To assess how EntF forms three ester linkages and cyclotrimerizes the covalent acyl enzyme DHB-Ser-S-PCP (peptidyl carrier protein) intermediate, we mutated residues of the proposed catalytic Ser-His-Asp triad of the thioesterase (TE) domain. RESULTS: The Ser1138-->Cys mutant (kcat decreased 1000-fold compared with wild-type EntF) releases both enterobactin (75%) and linear (DHB-Ser)2 dimer (25%) as products. The His 1271-->Ala mutant (kcat decreased 10,000-fold compared with wild-type EntF) releases only enterobactin, but accumulates both DHB-Ser-O-TE and (DHB-Ser)2-O-TE acyl enzyme intermediates. Electrospray ionization and Fourier transform mass spectrometry of proteolytic digests were used to analyze the intermediates. CONCLUSIONS: These results establish that the TE domain of EntF is both a cyclotrimerizing lactone synthetase and an elongation catalyst for ester-bond formation between covalently tethered DHB-Ser moieties, a new function for chain-termination TE domains found at the carboxyl termini of multimodular NRPSs and polyketide synthases.

Substrate recognition by class I lysyl-tRNA synthetases: a molecular basis for gene displacement.

Lysyl-tRNA synthetases (LysRSs) are unique amongst the aminoacyl-tRNA synthetases in being composed of unrelated class I and class II enzymes. To allow direct comparison between the two types of LysRS, substrate recognition by class I LysRSs was examined. Genes encoding both an archaeal and a bacterial class I enzyme were able to rescue an Escherichia coli strain deficient in LysRS, indicating their ability to functionally substitute for a class II LysRS in vivo. In vitro characterization showed lysine activation and recognition to be tRNA-dependent, an attribute of several class I, but not class II, aminoacyl-tRNA synthetases. Examination of tRNA recognition showed that class I LysRSs recognize the same elements in tRNALys as their class II counterparts, namely the discriminator base (N73) and the anticodon. This sequence-specific recognition of the same nucleotides in tRNALys by the two unrelated types of enzyme suggests that tRNALys predates at least one of the LysRSs in the evolution of the translational apparatus. The only observed variation in recognition was that the G2.U71 wobble pair of spirochete tRNALys acts as antideterminant for class II LysRS but does not alter class I enzyme recognition. This difference in tRNA recognition strongly favors the use of a class I-type enzyme to aminoacylate particular tRNALys species and provides a molecular basis for the observed displacement of class II by class I LysRSs in certain bacteria.