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Biomedical subjects

Peter F Leadlay

Publications and source records attributed to Peter F Leadlay.

At least 19 recordsLinked to original sources
PubMed2006

Separation of anti-angiogenic and cytotoxic activities of borrelidin by modification at the C17 side chain.

A set of novel borrelidin analogues have been prepared by precursor-directed biosynthesis. Structure-activity relationship analysis suggests that steric structural arrangement within the C17 side chain is important for differentiating cytotoxic and anti-angiogenic activities. A C17-cyclobutyl analogue 3 was found to have markedly increased selectivity for in vitro angiogenesis inhibition over cytotoxicity and is therefore potentially useful as an anticancer agent.

Angiogenesis Inhibitors↗
PubMed2006

Evidence that a novel thioesterase is responsible for polyketide chain release during biosynthesis of the polyether ionophore monensin.

Polyether ionophores, such as monensin A, are known to be biosynthesised, like many other antibiotic polyketides, on giant modular polyketide synthases (PKSs), but the intermediates and enzymes involved in the subsequent steps of oxidative cyclisation remain undefined. In particular there has been no agreement on the mechanism and timing of the final polyketide chain release. We now report evidence that MonCII from the monensin biosynthetic gene cluster in Streptomyces cinnamonensis, which was previously thought to be an epoxide hydrolase, is a novel thioesterase that belongs to the alpha/beta-hydrolase structural family and might catalyse this step. Purified recombinant MonCII was found to hydrolyse several thioester substrates, including an N-acetylcysteamine thioester derivative of monensin A. Further, incubation with a hallmark inhibitor of such enzymes, phenylmethanesulfonyl fluoride, led to inhibition of the thioesterase activity and to the accumulation of an acylated form of MonCII. These findings require a reassessment of the role of other enzymes implicated in the late stages of polyether ionophore biosynthesis.

Amino Acid Sequence↗
PubMed2006

High-throughput mutagenesis to evaluate models of stereochemical control in ketoreductase domains from the erythromycin polyketide synthase.

Ketoreductase (KR) activities help determine the stereochemistry of the products of modular polyketide synthases (PKSs). For example, domains eryKR(1) and eryKR(2), contained, respectively, in the first and second extension modules of the erythromycin-producing PKS, reduce 3-ketoacyl-thioester intermediates with opposite stereospecificity. Amino acid motifs that correlate with stereochemical outcome have been identified in KRs. We have used saturation mutagenesis of these motifs in eryKR(1) and eryKR(2), and a microplate-based screen of such mutants for activity against (9R, S)-trans-1-decalone, to identify candidate enzymes potentially altered in stereocontrol. Active mutants were reassayed with (2R, S)-2-methyl-3-oxopentanoic acid N-acetylcysteamine thioester, and the alcohol products were analyzed by chiral HPLC. Variant enzymes were found with either altered substrate selectivity for the (2R) or (2S) substrate or altered stereospecificity of reduction, or both, further highlighting the importance of these motifs in stereochemical control.

Amino Acid Sequence↗
PubMed2006

Directed mutagenesis alters the stereochemistry of catalysis by isolated ketoreductase domains from the erythromycin polyketide synthase.

The ketoreductase (KR) domains eryKR(1) and eryKR(2) from the erythromycin-producing polyketide synthase (PKS) reduce 3-ketoacyl-thioester intermediates with opposite stereospecificity. Modeling of eryKR(1) and eryKR(2) showed that conserved amino acids previously correlated with production of alternative alcohol configurations lie in the active site. eryKR(1) domains mutated at these positions showed an altered stereochemical outcome in reduction of (2R, S)-2-methyl-3-oxopentanoic acid N-acetylcysteamine thioester. The wild-type eryKR(1) domain exclusively gave the (2S, 3R)-3-hydroxy-2-methylpentanoic acid N-acetylcysteamine thioester, while the double mutant (F141W, P144G) gave only the (2S, 3S) isomer, a switch of the alcohol stereochemistry. Mutation of the eryKR(2) domain, in contrast, greatly increased the proportion of the wild-type (2R, 3S)-alcohol product. These data confirm the role of key residues in stereocontrol and suggest an additional way to make rational alterations in polyketide antibiotic structure.

Amino Acid Sequence↗
PubMed2006

Evidence for the role of the monB genes in polyether ring formation during monensin biosynthesis.

Ionophoric polyethers are produced by the exquisitely stereoselective oxidative cyclization of a linear polyketide, probably via a triepoxide intermediate. We report here that deletion of either or both of the monBI and monBII genes from the monensin biosynthetic gene cluster gave strains that produced, in place of monensins A and B, a mixture of C-3-demethylmonensins and a number of minor components, including C-9-epi-monensin A. All the minor components were efficiently converted into monensins by subsequent acid treatment. These data strongly suggest that epoxide ring opening and concomitant polyether ring formation are catalyzed by the MonB enzymes, rather than by the enzyme MonCII as previously thought. Consistent with this, homology modeling shows that the structure of MonB-type enzymes closely resembles the recently determined structure of limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis.

Amino Acid Sequence↗
PubMed2006

The gene cluster for fluorometabolite biosynthesis in Streptomyces cattleya: a thioesterase confers resistance to fluoroacetyl-coenzyme A.

A genomic library of Streptomyces cattleya was screened to isolate a gene cluster encoding enzymes responsible for the production of fluorine-containing metabolites. In addition to the previously described fluorinase FlA which catalyzes the formation of 5'-fluoro-5'-deoxyadenosine from S-adenosylmethionine and fluoride, 11 other putative open reading frames have been identified. Three of the proteins encoded by these genes have been characterized. FlB was determined to be the second enzyme in the pathway, catalyzing the phosphorolytic cleavage of 5'-fluoro-5'-deoxyadenosine to produce 5-fluoro-5-deoxy-D-ribose-1-phosphate. The enzyme FlI was found to be an S-adenosylhomocysteine hydrolase, which may act to relieve S-adenosylhomocysteine inhibition of the fluorinase. Finally, flK encodes a thioesterase which catalyzes the selective breakdown of fluoroacetyl-CoA but not acetyl-CoA, suggesting that it provides the producing strain with a mechanism for resistance to fluoroacetate.

Acetyl Coenzyme A↗
PubMed2006

Engineering of the spinosyn PKS: directing starter unit incorporation.

The spinosyns are a family of potent and highly selective insect control agents that display a favorable environmental profile. As some regions of the spinosyn molecule are recalcitrant to chemical modification, a targeted genetic approach was carried out to generate new analogues. The polyketide synthase (PKS) loading modules from the avermectin PKS of Streptomyces avermitilis and the erythromcyin PKS of Saccharopolyspora erythraea were each used to replace the spinosyn PKS loading module. Both of the resulting strains containing hybrid PKS pathways produced the anticipated spinosyn analogues. Supplementation of the culture media with a range of exogenous carboxylic acids led to the successful incorporation of these novel elements to yield further novel spinosyn molecules, some of which demonstrated potent and new insecticidal activities. Furthermore, it has been demonstrated that semisynthesis of such novel metabolites can then be used to generate active analogues, demonstrating the effectiveness of utilizing these complementary methods to search the chemical space around this template.

Amino Acid Sequence↗
PubMed2006

Organization of the biosynthetic gene cluster in Streptomyces sp. DSM 4137 for the novel neuroprotectant polyketide meridamycin.

Meridamycin is a non-immunosuppressant, FKBP-binding macrocyclic polyketide, which has major potential as a neuroprotectant in a range of neurodegenerative disorders including dementia, Parkinson's disease and ischaemic stroke. A biosynthetic cluster predicted to encode biosynthesis of meridamycin was cloned from the prolific secondary-metabolite-producing strain Streptomyces sp. DSM 4137, not previously known to produce this compound, and specific gene deletion was used to confirm the role of this cluster in the biosynthesis of meridamycin. The meridamycin modular polyketide synthase consists of 14 extension modules distributed between three giant multienzyme proteins. The terminal module is flanked by a highly unusual cytochrome P450-like domain. The characterization of the meridamycin biosynthetic locus in this readily manipulated streptomycete species opens the way to the engineering of new, altered meridamycins of potential therapeutic importance.

Bacterial Proteins↗
PubMed2005

Molecular basis of Celmer's rules: stereochemistry of catalysis by isolated ketoreductase domains from modular polyketide synthases.

A system is reported for the recombinant expression of individual ketoreductase (KR) domains from modular polyketide synthases (PKSs) and scrutiny of their intrinsic specificity and stereospecificity toward surrogate diketide substrates. The eryKR(1) and the tylKR(1) domains, derived from the first extension module of the erythromycin PKS and the tylosin PKS, respectively, both catalyzed reduction of (2R, S)-2-methyl-3-oxopentanoic acid N-acetylcysteamine thioester, with complete stereoselectivity and stereospecificity, even though the substrate is not tethered to an acyl carrier protein or an intact PKS multienzyme. In contrast, and to varying degrees, the isolated enzymes eryKR(2), eryKR(5), and eryKR(6) exercised poorer control over substrate selection and the stereochemical course of ketoreduction. These data, together with modeling of diketide binding to KR(1) and KR(2), demonstrate the fine energetic balance between alternative modes of presentation of ketoacylthioester substrates to KR active sites.

Binding Sites↗
PubMed2005

Combinatorial biosynthesis of reduced polyketides.

The bacterial multienzyme polyketide synthases (PKSs) produce a diverse array of products that have been developed into medicines, including antibiotics and anticancer agents. The modular genetic architecture of these PKSs suggests that it might be possible to engineer the enzymes to produce novel drug candidates, a strategy known as 'combinatorial biosynthesis'. So far, directed engineering of modular PKSs has resulted in the production of more than 200 new polyketides, but key challenges remain before the potential of combinatorial biosynthesis can be fully realized.

Bacteria↗
PubMed2005

Organization of the biosynthetic gene cluster for the macrolide concanamycin A in Streptomyces neyagawaensis ATCC 27449.

The macrolide antibiotic concanamycin A has been identified as an exceptionally potent inhibitor of the vacuolar (V-type) ATPase. Such compounds have been mooted as the basis of a potential drug treatment for osteoporosis, since the V-ATPase is involved in the osteoclast-mediated bone resorption that underlies this common condition. To enable combinatorial engineering of altered concanamycins, the biosynthetic gene cluster governing the biosynthesis of concanamycin A has been cloned from Streptomyces neyagawaensis and shown to span a region of over 100 kbp of contiguous DNA. An efficient transformation system has been developed for S. neyagawaensis and used to demonstrate the role of the cloned locus in the formation of concanamycin A. Sequence analysis of the 28 ORFs in the region has revealed key features of the biosynthetic pathway, in particular the biosynthetic origin of portions of the backbone, which arise from the unusual polyketide building blocks ethylmalonyl-CoA and methoxymalonyl-ACP, and the origin of the pendant deoxysugar moiety 4'-O-carbamoyl-2'-deoxyrhamnose, as well as the presence of a modular polyketide synthase (PKS) encoded by six giant ORFs. Examination of the methoxymalonyl-specific acyltransferase (AT) domains has led to recognition of an amino acid sequence motif which can be used to distinguish methylmalonyl-CoA- from methoxymalonyl-ACP-specific AT domains in natural PKSs.

Acyl Carrier Protein↗
PubMed2005

Chain initiation on type I modular polyketide synthases revealed by limited proteolysis and ion-trap mass spectrometry.

Limited proteolysis in combination with liquid chromatography-ion trap mass spectrometry (LC-MS) was used to analyze engineered or natural proteins derived from a type I modular polyketide synthase (PKS), the 6-deoxyerythronolide B synthase (DEBS), and comprising either the first two extension modules linked to the chain-terminating thioesterase (TE) (DEBS1-TE); or the last two extension modules (DEBS3) or the first extension module linked to TE (diketide synthase, DKS). Functional domains were released by controlled proteolysis, and the exact boundaries of released domains were obtained through mass spectrometry and N-terminal sequencing analysis. The acyltransferase-acyl carrier protein required for chain initiation (AT(L)-ACP(L)), was released as a didomain from both DEBS1-TE and DKS, as well as the off-loading TE as a didomain with the adjacent ACP. Mass spectrometry was used successfully to monitor in detail both the release of individual domains, and the patterns of acylation of both intact and digested DKS when either propionyl-CoA or n-butyryl-CoA were used as initiation substrates. In particular, both loading domains and the ketosynthase domain of the first extension module (KS1) were directly observed to be simultaneously primed. The widely available and simple MS methodology used here offers a convenient approach to the proteolytic mapping of PKS multienzymes and to the direct monitoring of enzyme-bound intermediates.

Acyl Coenzyme A↗