Intermediates in monensin biosynthesis: A late step in biosynthesis of the polyether ionophore monensin is crucial for the integrity of cation binding.

Polyether antibiotics such as monensin are biosynthesised via a cascade of directed ring expansions operating on a putative polyepoxide precursor. The resulting structures containing fused cyclic ethers and a lipophilic backbone can form strong ionophoric complexes with certain metal cations. In this work, we demonstrate for monensin biosynthesis that, as well as ether formation, a late-stage hydroxylation step is crucial for the correct formation of the sodium monensin complex. We have investigated the last two steps in monensin biosynthesis, namely hydroxylation catalysed by the P450 monooxygenase MonD and O-methylation catalysed by the methyl-transferase MonE. The corresponding genes were deleted in-frame in a monensin-overproducing strain of Streptomyces cinnamonensis. The mutants produced the expected monensin derivatives in excellent yields (ΔmonD: 1.13 g L(-1) dehydroxymonensin; ΔmonE: 0.50 g L(-1) demethylmonensin; and double mutant ΔmonDΔmonE: 0.34 g L(-1) dehydroxydemethylmonensin). Single crystals were obtained from purified fractions of dehydroxymonensin and demethylmonensin. X-ray structure analysis revealed that the conformation of sodium dimethylmonensin is very similar to that of sodium monensin. In contrast, the coordination of the sodium ion is significantly different in the sodium dehydroxymonensin complex. This shows that the final constitution of the sodium monensin complex requires this tailoring step as well as polyether formation.

Structural basis for the activity and substrate specificity of fluoroacetyl-CoA thioesterase FlK.

The thioesterase FlK from the fluoroacetate-producing Streptomyces cattleya catalyzes the hydrolysis of fluoroacetyl-coenzyme A. This provides an effective self-defense mechanism, preventing any fluoroacetyl-coenzyme A formed from being further metabolized to 4-hydroxy-trans-aconitate, a lethal inhibitor of the tricarboxylic acid cycle. Remarkably, FlK does not accept acetyl-coenzyme A as a substrate. Crystal structure analysis shows that FlK forms a dimer, in which each subunit adopts a hot dog fold as observed for type II thioesterases. Unlike other type II thioesterases, which invariably utilize either an aspartate or a glutamate as catalytic base, we show by site-directed mutagenesis and crystallography that FlK employs a catalytic triad composed of Thr(42), His(76), and a water molecule, analogous to the Ser/Cys-His-acid triad of type I thioesterases. Structural comparison of FlK complexed with various substrate analogues suggests that the interaction between the fluorine of the substrate and the side chain of Arg(120) located opposite to the catalytic triad is essential for correct coordination of the substrate at the active site and therefore accounts for the substrate specificity.

Synthetic chain terminators off-load intermediates from a type I polyketide synthase.

Modular biocatalysis is responsible for the generation of countless bioactive products and its mining remains a major focus for drug discovery purposes. One of the enduring hurdles is the isolation of biosynthetic intermediates in a readily-analysed form. We prepared a series of nonhydrolysable pantetheine and N-acetyl cysteamine mimics of the natural (methyl)malonyl extender units recruited for polyketide formation. Using these analogues as competitive substrates, we were able to trap and off-load diketide and triketide species directly from an in vitro reconstituted type I polyketide synthase, the 6-deoxyerythronolide B synthase 3 (DEBS3). The putative intermediates, which were extracted in organic solvent and characterised by LC-HR-ESI-MS, are the first of their kind and prove that small-molecule chain terminators can be used as convenient probes of the biosynthetic process.

Malonyl carba(dethia)- and malonyl oxa(dethia)-coenzyme A as tools for trapping polyketide intermediates.

In order to study intermediates in polyketide biosynthesis two nonhydrolyzable malonyl coenzyme A analogues were synthesised by a chemoenzymatic route. In these analogues the sulfur atom of CoA was replaced either by a methylene group (carbadethia analogue) or by an oxygen atom (oxadethia analogue). These malonyl-CoA analogues were found to compete with the natural extender unit malonyl-CoA and to trap intermediates from stilbene synthase, a type III polyketide synthase (PKS). From the reaction of stilbene synthase with its natural phenylpropanoid substrates, diketide, triketide and tetraketide species were successfully off-loaded and characterised by LC-MS. Moreover, the reactivity of the nonhydrolyzable analogues offers insights into the flexibility of substrate alignment in the PKS active site for efficient malonyl decarboxylation and condensation.

Chimeric glycosyltransferases for the generation of hybrid glycopeptides.

Glycodiversification, an invaluable tool for generating biochemical diversity, can be catalyzed by glycosyltransferases, which attach activated sugar "donors" onto "acceptor" molecules. However, many glycosyltransferases can tolerate only minor modifications to their native substrates, thus making them unsuitable tools for current glycodiversification strategies. Here we report the production of functional chimeric glycosyltransferases by mixing and matching the N- and C-terminal domains of glycopeptide glycosyltransferases. Using this method we have generated hybrid glycopeptides and have demonstrated that domain swapping can result in a predictable switch of substrate specificity, illustrating that N- and C-terminal domains predominantly dictate acceptor and donor specificity, respectively. The determination of the structure of a chimera in complex with a sugar donor analog shows that almost all sugar-glycosyltransferase binding interactions occur in the C-terminal domain.

Possible origin of electronic effects in Rh(I)-catalyzed enantioselective hydrogenation.

Reducing the electron density of ligands switches the regioselectivity of Rh(I)-catalyzed hydrometalation. A reversal of the sense of chiral induction was also observed when chiral ligands are electronically tuned in the same manner. The combined data provide an alternative rationale for the electronic effects often observed in asymmetric hydrogenation.

Identification of a pentaketide stilbene produced by a type III polyketide synthase from Pinus sylvestris and characterisation of free coenzyme A intermediates.

The stilbene synthase (STS) from Scots pine (Pinus sylvestris), which is a type III polyketide synthase, produces the well known tetraketide resveratrol from coumaroyl-CoA and three molecules of malonyl-CoA. The same stilbene synthase, however, also generates the previously unknown pentaketide 2-malonylresveratrol from coumaroyl-CoA and four molecules of malonyl-CoA; this indicates that the enzyme does not precisely control the number of condensations leading to diverse products. Tetraketide- and pentaketide-CoA intermediates of the STS were identified by LC-MS/MS; this suggests that CoA-bound polyketide intermediates are involved in the product formation of type III polyketide synthases.

Analysis of specific mutants in the lasalocid gene cluster: evidence for enzymatic catalysis of a disfavoured polyether ring closure.

Lasalocid is a highly atypical polyether ionophoric antibiotic, firstly because it contains a type of aromatic ring normally associated with fungal polyketides, and secondly because the formation of its tetrahydropyran ring appears to contravene Baldwin's rules, which predict the kinetically preferred routes for cyclisation reactions in organic chemistry. The lasalocid biosynthetic gene cluster has been cloned from Streptomyces lasaliensis, and the las locus (73,533 bp) was found to contain seven modular polyketide synthase (PKS) genes, including all the activities necessary for the synthesis of the aromatic moiety. Specific deletion from the gene cluster of the flanking lasC gene, which is predicted to encode a flavin-linked epoxidase, abolished production both of lasalocid and of the minor cometabolite iso-lasalocid without leading to accumulation of an identifiable intermediate; this suggests that oxidative cyclisation to form the polyether rings takes place on the PKS before release of the full-length polyketide product. Meanwhile, a mutant in which the adjacent epoxide hydrolase lasB had been deleted produced iso-lasalocid only. Iso-lasalocid differs from lasalocid in the replacement of the tetrahydropyran ring by a tetrohydrofuran ring and represents the kinetically favoured product of cyclisation. The LasB epoxide hydrolase is therefore directly implicated in control of the stereochemical course of polyether ring formation during lasalocid biosynthesis.

The neomycin biosynthetic gene cluster of Streptomyces fradiae NCIMB 8233: genetic and biochemical evidence for the roles of two glycosyltransferases and a deacetylase.

An efficient protocol has been developed for the genetic manipulation of Streptomyces fradiae NCIMB 8233, which produces the 2-deoxystreptamine (2-DOS)-containing aminoglycoside antibiotic neomycin. This has allowed the in vivo analysis of the respective roles of the glycosyltransferases Neo8 and Neo15, and of the deacetylase Neo16 in neomycin biosynthesis. Specific deletion of each of the neo8, neo15 and neo16 genes confirmed that they are all essential for neomycin biosynthesis. The pattern of metabolites produced by feeding putative pathway intermediates to these mutants provided unambiguous support for a scheme in which Neo8 and Neo15, whose three-dimensional structures are predicted to be highly similar, have distinct roles: Neo8 catalyses transfer of N-acetylglucosamine to 2-DOS early in the pathway, while Neo15 catalyses transfer of the same aminosugar to ribostamycin later in the pathway. The in vitro substrate specificity of Neo15, purified from recombinant Escherichia coli, was fully consistent with these findings. The in vitro activity of Neo16, the only deacetylase so far recognised in the neo gene cluster, showed that it is capable of acting in tandem with both Neo8 and Neo15 as previously proposed. However, the deacetylation of N-acetylglucosaminylribostamycin was still observed in a strain deleted of the neo16 gene and fed with suitable pathway precursors, providing evidence for the existence of a second enzyme in S. fradiae with this activity.

Chemoenzymatic acylation of aminoglycoside antibiotics.

The chemoenzymatic installation of the clinically valuable (S)-4-amino-2-hydroxybutyryl side chain onto a number of 2-deoxystreptamine-containing aminoglycosides is described using the purified Bacillus circulans biosynthetic enzymes BtrH and BtrG in combination with a synthetic acyl-SNAC surrogate substrate.

The role of cep15 in the biosynthesis of chloroeremomycin: reactivation of an ancestral catalytic function.

The gene clusters of several glycopeptides contain genes that encode COG2120 domain zinc-dependent N-acetylglucosaminyl deacetylases. Recently, a COG2120 protein encoded in the chloroeremomycin gene cluster, Cep15, has been postulated to possess nucleotidyltransferase activity. Here, we demonstrate that Cep15 possesses no catalytic activity and does not have a clear role in chloroeremomycin biosynthesis. This result strongly suggests that cep15 and bal2 are evolutionary artifacts and may be pseudogenes. Comparative sequence analysis with the closely related active Orf2* deacetylase (teicoplanin biosynthesis) reveals an asparagine in place of a metal-binding histidine in the "pseudo-active site" of Cep15. Substitution of this histidine by asparagine in Orf2* abolishes deacetylase activity. Remarkably, the Cep15 N164H mutant is an active deacetylase. To our knowledge, this is the first example of reactivating an ancestral enzymatic role for a bacterial protein by point mutagenesis.

Analysis of the tetronomycin gene cluster: insights into the biosynthesis of a polyether tetronate antibiotic.

The biosynthetic gene cluster for tetronomycin (TMN), a polyether ionophoric antibiotic that contains four different types of ring, including the distinctive tetronic acid moiety, has been cloned from Streptomyces sp. NRRL11266. The sequenced tmn locus (113 234 bp) contains six modular polyketide synthase (PKS) genes and a further 27 open-reading frames. Based on sequence comparison to related biosynthetic gene clusters, the majority of these can be assigned a plausible role in TMN biosynthesis. The identity of the cluster, and the requirement for a number of individual genes, especially those hypothesised to contribute a glycerate unit to the formation of the tetronate ring, were confirmed by specific gene disruption. However, two large genes that are predicted to encode together a multifunctional PKS of a highly unusual type seem not to be involved in this pathway since deletion of one of them did not alter tetronomycin production. Unlike previously characterised polyether PKS systems, oxidative cyclisation appears to take place on the modular PKS rather than after transfer to a separate carrier protein, while tetronate ring formation and concomitant chain release share common mechanistic features with spirotetronate biosynthesis.

Covalent linkage mediates communication between ACP and TE domains in modular polyketide synthases.

Polyketide natural products such as erythromycin A and epothilone are assembled on multienzyme polyketide synthases (PKSs), which consist of modular sets of protein domains. Within these type I systems, the fidelity of biosynthesis depends on the programmed interaction among the multiple domains within each module, centered around the acyl carrier protein (ACP). A detailed understanding of interdomain communication will therefore be vital for attempts to reprogram these pathways by genetic engineering. We report here that the interaction between a representative ACP domain and its downstream thioesterase (TE) is mediated largely by covalent tethering through a short "linker" region, with only a minor energetic contribution from protein-protein molecular recognition. This finding helps explain in part the empirical observation that TE domains can function out of their normal context in engineered assembly lines, and supports the view that overall PKS architecture may dictate at least a subset of interdomain interactions.

Glyceryl-S-acyl carrier protein as an intermediate in the biosynthesis of tetronate antibiotics.

The biosynthetic pathway to the unusual tetronate ring of certain polyketide natural products, including the antibiotics abyssomicin and tetronomycin (TMN) and the antitumour compound chlorothricin (CHL), is presently unknown. The gene clusters governing chlorothricin and tetronomycin biosynthesis both contain a gene encoding an atypical member of the FkbH family of enzymes, which has previously been shown to synthesise glyceryl-S-acyl carrier protein (ACP) as the first step in production of unusual extender units for modular polyketide biosynthesis. We show here that purified recombinant FkbH-like protein, Tmn16, from the TMN gene cluster catalyses the efficient transfer of a glyceryl moiety from D-1,3-bisphosphoglycerate (1,3-BPG) to either of the dedicated ACPs, Tmn7a and ChlD2, to form glyceryl-S-ACP, which directly implicates this compound as an intermediate in tetronate biosynthesis as well. Neither Tmn16 nor Tmn7a produced glyceryl-S-ACP when incubated, respectively, with analogous ACP and FkbH-like proteins from a known extender-unit pathway; this indicates a highly selective channelling of glycolytic metabolites into tetronate biosynthesis.

Biosynthesis of butirosin: transfer and deprotection of the unique amino acid side chain.

Butirosin, an aminoglycoside antibiotic produced by Bacillus circulans, bears the unique (S)-4-amino-2-hydroxybutyrate (AHBA) side chain, which protects the antibiotic from several common resistance mechanisms. The AHBA side chain is advantageously incorporated into clinically valuable antibiotics such as amikacin and arbekacin by synthetic methods. Therefore, it is of significant interest to explore the biosynthetic origins of this useful moiety. We report here that the AHBA side chain of butirosin is transferred from the acyl carrier protein (ACP) BtrI to the parent aminoglycoside ribostamycin as a gamma-glutamylated dipeptide by the ACP:aminoglycoside acyltransferase BtrH. The protective gamma-glutamyl group is then cleaved by BtrG via an uncommon gamma-glutamyl cyclotransferase mechanism. The application of this pathway to the in vitro enzymatic production of novel AHBA-bearing aminoglycosides is explored with encouraging implications for the preparation of unnatural antibiotics via directed biosynthesis.

Elaboration of neosamine rings in the biosynthesis of neomycin and butirosin.

The proteins Neo-11 and Neo-18 encoded in the neomycin gene cluster (neo) of Streptomyces fradiae NCIMB 8233 have been characterized as glucosaminyl-6'-oxidase and 6'-oxoglucosaminyl:L-glutamate aminotransferase, respectively. The joint activity of Neo-11 and Neo-18 is responsible for the conversion of paromamine to neamine in the biosynthetic pathway of neomycin through a mechanism of FAD-dependent dehydrogenation followed by a pyridoxal-5'-phosphate-mediated transamination. Neo-18 is also shown to catalyze deamination at C-6''' of neomycin, thus suggesting bifunctional roles of the two enzymes in the formation of both neosamine rings of neomycin. The product of the btrB gene, a homologue of neo-18 in the butirosin biosynthetic gene cluster (btr) in Bacillus circulans, exhibits the same activity as Neo-18; this indicates that there is a similar reaction sequence in both butirosin and neomycin biosynthesis.