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Biomedical subjects

P F Leadlay

Publications and source records attributed to P F Leadlay.

At least 55 records · Page 3Linked to original sources
PubMed1996

Organization of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of the enzymatic domains in the modular polyketide synthase.

The three giant multifunctional polypeptides of the rapamycin (Rp)-producing polyketide synthase (RAPS1, RAPS2 and RAPS3) have recently been shown to contain 14 separate sets, or modules, of enzyme activities, each module catalysing a specific round of polyketide chain extension. Detailed sequence comparison between these protein modules has allowed further characterisation of aa that may be important in catalysis or specificity. The acyl-carrier protein (ACP), beta-ketoacyl-ACP synthase (KS) and acyltransferase (AT) domains (the core domains) have an extremely high degree of mutual sequence homology. The KS domains in particular are almost perfect repeats over their entire length. Module 14 shows the least homology and is unique in possessing only core domains. The enoyl reductase (ER), beta-ketoacyl-ACP reductase (KR) and dehydratase (DH) domains are present even in certain modules where they are not apparently required. Four DH domains can be recognised as inactive by characteristic deletions in active site sequences, but for two others, and for KR and ER in module 3, the sequence is not distinguishable from that of active counterparts in other modules. The N terminus of RAPS1 contains a novel coenzyme A ligase (CL) domain that activates and attaches the shikimate-derived starter unit, and an ER activity that may modify the starter unit after attachment. The sequence comparison has revealed the surprisingly high sequence similarity between inter-domain 'linker' regions, and also a potential amphipathic helix at the N terminus of each multienzyme subunit which may promote dimerisation into active species.

Amino Acid Sequence↗
PubMed1996

Homology modeling of human methylmalonyl-CoA mutase: a structural basis for point mutations causing methylmalonic aciduria.

Point mutations in the human gene encoding coenzyme B12 (adenosylcobalamin)-dependent methylmalonyl-CoA mutase give rise to an inherited disorder of propionic acid metabolism termed mut methylmalonic aciduria. Almost all such mutations alter amino acids in the homodimeric human enzyme that are identical to residues in the catalytic alpha-subunit of the heterodimeric methylmalonyl-CoA mutase from the bacterium Propionibacterium shermanii, to which the mature human enzyme shows an overall 65% sequence identity. To explore how specific mutations might cause the observed clinical phenotype, 12 known mutations were mapped onto a three-dimensional homology model of the subunit of the human enzyme, generated using the program MODELLER on the basis of the recently published 2.0 A X-ray crystal structure of the P. shermanii methylmalonyl-CoA mutase. Eight mutations are found in the C-terminal B12-binding domain, of which 4 (G623R, G626C, G630E, G703R) are in direct contact with the corrin and are clustered around the histidine ligand (H627) provided by the protein to coordinate the cobalt atom of the B12 cofactor. Introduction of a side chain, particularly one that is charged, at any of these positions is expected to disrupt the flavodoxin-like fold and severely impair its binding of B12. Mutation at either of two other highly conserved glycine residues in this domain (G648D, G717V) also disrupts critical elements in the fold as would the introduction of an additional positive charge in the mutation H678R. Mutation of an arginine in a solvent-exposed loop to a hydrophobic residue (R694W) is also pathogenic. The remaining mutations have been mapped to the N-terminal region of the mutase, two of which introduce a buried, uncompensated charge, either near the subunit interface (A377E), or near the narrow channel through which acyl-CoA esters gain access to the active site (W105R). The extreme N-terminus of methylmalonyl-CoA mutase is predicted to make extensive contacts with the other subunit, and a mutant in this region (R93H) may prevent the correct assembly of the dimer.

Amino Acid Metabolism, Inborn Errors↗
PubMed1996

A hybrid modular polyketide synthase obtained by domain swapping.

BACKGROUND: Modular polyketide synthases govern the synthesis of a number of medically important antibiotics, and there is therefore great interest in understanding how genetic manipulation may be used to produce hybrid synthases that might synthesize novel polyketides. In particular, we aimed to show whether an individual domain can be replaced by a comparable domain from a different polyketide synthase to form a functional hybrid enzyme. To simplify the analysis, we have used our previously-developed model system DEBS1-TE, consisting of the first two chain-extension modules of the erythromycin-producing polyketide synthase of Saccharopolyspora erythraea. RESULTS: We show here that replacing the entire acyltransferase (AT) domain from module 1 of DEBS1-TE by the AT domain from module 2 of the rapamycin-producing polyketide synthase leads, as predicted, to the synthesis of two novel triketide lactones in good yield, in place of the two lactones produced by DEBS1-TE. Both of the novel products specifically lack a methyl group at C-4 of the lactone ring. CONCLUSIONS: Although the AT domain is a core structural domain of a modular polyketide synthase, it has been swapped to generate a truly hybrid multienzyme with a rationally altered specificity of chain extension. Identical manipulations carried out on known polyketide antibiotics might therefore generate families of potentially useful analogues that are inaccessible by chemical synthesis. These results also encourage the belief that other domains may be similarly swapped.

Amino Acid Sequence↗
PubMed1996

Evidence for a double-helical structure for modular polyketide synthases.

Modular polyketide synthases are multienzymes responsible for the biosynthesis of a large number of clinically important natural products. They contain multiple sets, or modules, of enzymatic activities, distributed between a few giant multienzymes and there is one module for every successive cycle of polyketide chain extension. We show here that each multienzyme in a typical modular polyketide synthase forms a (possibly helical) parallel dimer, and that each pair of identical modules interacts closely across the dimer interface. Such an arrangement would allow identical modules to share active sites for chain extension, and thus to function independently of flanking modules, which would have important implications both for mechanisms of evolution of polyketide synthases and for their future genetic engineering.

Amino Acid Sequence↗
PubMed1995

Divergent sequence motifs correlated with the substrate specificity of (methyl)malonyl-CoA:acyl carrier protein transacylase domains in modular polyketide synthases.

The amino acid sequences of a large number of polyketide synthase domains that catalyse the transacylation of either methylmalonyl-CoA or malonyl-CoA onto acyl carrier protein (ACP) have been compared. Regions were identified in which the acyltransferase sequences diverged according to whether they were specific for malonyl-CoA or methylmalonyl-CoA. These differences are sufficiently clear to allow unambiguous assignment of newly-sequenced acyltransferase domains in modular polyketide synthases. Comparison with the recently-determined structure of the malonyltransferase from Escherichia coli fatty acid synthase showed that the divergent region thus identified lies near the acyltransferase active site, though not close enough to make direct contact with bound substrate.

Acyl Carrier Protein↗
PubMed1995

Repositioning of a domain in a modular polyketide synthase to promote specific chain cleavage.

Macrocyclic polyketides exhibit an impressive range of medically useful activities, and there is great interest in manipulating the genes that govern their synthesis. The 6-deoxyerythronolide B synthase (DEBS) of Saccharopolyspora erythraea, which synthesizes the aglycone core of the antibiotic erythromycin A, has been modified by repositioning of a chain-terminating cyclase domain to the carboxyl-terminus of DEBS1, the multienzyme that catalyzes the first two rounds of polyketide chain extension. The resulting mutant markedly accelerates formation of the predicted triketide lactone, compared to a control in which the repositioned domain is inactive. Repositioning of the cyclase should be generally useful for redirecting polyketide synthesis to obtain polyketides of specified chain lengths.

Base Sequence↗
PubMed1995

Polyketide synthesis in vitro on a modular polyketide synthase.

BACKGROUND: The 6-deoxyerythronolide B synthase (DEBS) of Saccharopolyspora erythraea, which synthesizes the aglycone core of the antibiotic erythromycin A, contains some 30 active sites distributed between three multienzyme polypeptides (designated DEBS1-3). This complexity has hitherto frustrated mechanistic analysis of such enzymes. We previously produced a mutant strain of S. erythraea in which the chain-terminating cyclase domain (TE) is fused to the carboxyl-terminus of DEBS1, the multienzyme that catalyzes the first two rounds of polyketide chain extension in S. erythraea. This mutant strain produces triketide lactone in vivo. We set out to purify the chimaeric enzyme and to determine its activity in vitro. RESULTS: The purified DEBS1-TE multienzyme catalyzes synthesis of triketide lactones in vitro. The synthase specifically uses the (2S)-isomer of methylmalonyl-CoA, as previously proposed, but has a more relaxed specificity for the starter unit than in vivo. CONCLUSIONS: We have obtained a purified polyketide synthase system, derived from DEBS, which retains catalytic activity. This approach opens the way for mechanistic and structural analyses of active multienzymes derived from any modular polyketide synthase.

Cell-Free System↗
PubMed1994

Limited proteolysis and active-site studies of the first multienzyme component of the erythromycin-producing polyketide synthase.

The domain structure of the 6-deoxyerythronolide B synthase 1 component of the erythromycin-producing polyketide synthase from Saccharopolyspora erythraea has been investigated using limited proteolysis and active-site labeling. Trypsin, elastase, endoproteinase Glu-C, and endoproteinase Arg-C were used to cleave the multienzyme, and the sizes of the resulting fragments were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The location of fragments within the primary structure was established by N-terminal sequence analysis. The cleavage pattern followed domain boundaries previously predicted on the basis of sequence alignments, but many predicted interdomain regions were not cleaved, even under the harshest conditions used. Initial proteolysis generated three large fragments: an N-terminal fragment (about 60 kDa) housing an acyltransferase-acyl carrier protein di-domain; a central fragment (about 90 kDa) containing a ketosynthase-acyltransferase di-domain; and a C-terminal fragment (about 220 kDa) containing the remaining six domains of the multienzyme, including the third acyltransferase. The intact multienzyme behaves as a dimer of molecular mass 660 kDa on gel filtration; and the C-terminal fragment remains dimeric. However, the N-terminal and central fragments appear to be monomeric species. After proteolysis of the multienzyme, the N-terminal di-domain was found to be specifically labeled after incubation with [14C]propionyl-CoA, providing the first evidence for its proposed role as a "loading domain" for the propionate starter unit. In contrast, the other two fragments were specifically acylated by [14C]methylmalonyl-CoA, indicating that both the other two acyltransferases remain enzymatically active after proteolysis.

Amino Acid Sequence↗
PubMed1994

Stereospecific acyl transfers on the erythromycin-producing polyketide synthase.

During assembly of complex polyketide antibiotics like erythromycin A, molecular recognition by the multienzyme polyketide synthase controls the stereochemical outcome as each successive methylmalonyl-coenzyme A (CoA) extender unit is added. Acylation of the purified erythromycin-producing polyketide synthase has shown that all six acyltransferase domains have identical stereospecificity for their normal substrate, (2S)-methylmalonyl-CoA. In contrast, the configuration of the methyl-branched centers in the product, that are derived from (2S)-methylmalonyl-CoA, is different. Stereoselection during the chain building process must, therefore, involve additional epimerization steps.

Acetyl Coenzyme A↗
PubMed1994

An ABC-transporter from Streptomyces longisporoflavus confers resistance to the polyether-ionophore antibiotic tetronasin.

Streptomyces longisporoflavus produces the polyketide-polyether antibiotic, tetronasin, which acts as an ionophore and depolarizes the membrane of bacteria sensitive to the drug. A genomic library of S. longisporoflavus DNA was cloned in Streptomyces lividans and screened to identify tetronasin-resistance determinants. The inclusion of 0.2M NaCl in the growth medium with tetronasin markedly improved the sensitivity of the screen. Two different resistance determinants, designated tnrB (ptetR51) and tnrA (ptetR11) respectively, were identified. The determinant tnrB (ptetR51) but not tnrA (ptetR11), also conferred resistance to tetronasin when cloned into Streptomyces albus. The tnrB determinant was further localized, by subcloning, to a 2.8 kb KpnI fragment. DNA sequence analysis of this insert revealed one incomplete and two complete open reading frames (ORFs 1, 2 and 3). The deduced sequence of the gene product of ORF2 (TnrB2) revealed significant similarity to the ATP-binding domains of the ABC (ATP binding cassette) superfamily of transport-related proteins. The adjacent gene, ORF3, is translationally coupled to ORF2 and would encode a hydrophobic protein (TnrB3) with six transmembrane helices which probably constitutes the integral membrane component of the transporter. The mechanism of tetronasin resistance mediated by tnrB is probably an ATP-dependent efflux system.

Amino Acid Sequence↗
PubMed1993

The synthetic substrate succinyl(carbadethia)-CoA generates cob(II)alamin on adenosylcobalamin-dependent methylmalonyl-CoA mutase.

Succinyl(carbadethia)-coenzyme A, a synthetic substrate for adenosylcobalamin-dependent methylmalonyl-CoA mutase, has been prepared by a simplified procedure. When recombinant mutase was mixed with the synthetic substrate, the u.v./visible absorption spectrum of the bound cofactor changed rapidly to resemble that of cob(II)alamin, with an absorption maximum at 467 nm. Addition of the natural substrates, in contrast, produced only minor changes in the u.v./visible spectrum. The recent report of the generation of a complex e.p.r. spectrum on addition of substrate to the holo-methylmalonyl-CoA mutase was confirmed with the recombinant enzyme. The signals observed were stronger when the succinyl(carbadethia) analogue was used. Cobalt K-edge X-ray absorption spectroscopy confirmed that the addition of this analogue to holoenzyme leads to the generation of a cob(II)alamin-like species. These results strongly support the generation of cob(II)alamin during the 1,2-skeletal rearrangement catalysed by methylmalonyl-CoA mutase, as required if this enzyme follows the reaction pathway involving radical intermediates previously proposed for other adenosylcobalamin-dependent enzymes.

Acyl Coenzyme A↗
PubMed1993

Purification and separation of holo- and apo-forms of Saccharopolyspora erythraea acyl-carrier protein released from recombinant Escherichia coli by freezing and thawing.

Saccharopolyspora erythraea acyl-carrier protein, highly expressed from a T7-based expression plasmid in Escherichia coli, can be selectively released from the cells in near-quantitative yield by a single cycle of freezing and thawing in a neutral buffer. Electrospray mass spectrometry was used to confirm that the recombinant S. erythraea acyl-carrier protein over-expressed in E. coli is present predominantly as the holo-form, with variable amounts of apo-acyl-carrier protein, holo-acyl-carrier protein dimer and holo-acyl-carrier protein glutathione adduct. The holo- and apo-acyl-carrier proteins are both readily purified on a large scale from the freeze-thaw extracts and can be separated from one another by octyl-Sepharose chromatography. The holo-acyl-carrier protein obtained in this way was fully active in supporting the synthesis of acyl-acyl-carrier protein by extracts of S. erythraea.

Acyl Carrier Protein↗
PubMed1993

Heterologous expression in Escherichia coli of an intact multienzyme component of the erythromycin-producing polyketide synthase.

6-Deoxyerythronolide B synthase 3 (DEBS 3) is proposed to catalyse the fifth and sixth condensation cycles in the assembly of the polyketide 6-deoxyerythronolide B, the first isolatable intermediate in the biosynthesis of erythromycin A by Saccharopolyspora erythraea. The gene encoding DEBS 3 has previously been cloned and sequenced, and the deduced product is predicted to house nine fatty acid synthase-like activities on a 330-kDa polypeptide chain. The gene has been engineered into a pT-7-based expression system for over-expression in Escherichia coli. Recombinant DEBS 3 was found to constitute, after induction, 1-2% of soluble intracellular protein. DEBS 3 was purified from extracts of the recombinant E. coli to apparent homogeneity, and was found not to be modified by covalent attachment of the prosthetic group 4'-phosphopantetheine. Incubation with (R,S)-methylmalonyl-CoA, the presumed source of extension units for polyketide chain assembly, led to hydrolysis of the thioester, implying that the methylmalonyl-CoA:ACP acyltransferase domains in DEBS 3 are correctly folded and able to catalyse this side-reaction. During this reaction, DEBS 3 became transiently radiolabelled, consistent with the intermediacy of an acylenzyme. The native molecular mass of the protein by gel filtration chromatography was 668 kDa which corresponds either to a dimer or to a highly asymmetric monomer.

Acyl Coenzyme A↗
PubMed1992

Identification of DEBS 1, DEBS 2 and DEBS 3, the multienzyme polypeptides of the erythromycin-producing polyketide synthase from Saccharopolyspora erythraea.

The ery A region of the erythromycin biosynthetic gene cluster of Saccharopolyspora erythraea has previously been shown to contain three large open reading frames (ORFs) that encode the components of 6-deoxyerythronolide B synthase (DEBS). Polyclonal antibodies were raised against recombinant proteins obtained by overexpression of 3' regions of the ORF2 and ORF3 genes. In Western blotting experiments, each antiserum reacted strongly with a different high molecular weight protein in extracts of erythromycin-producing S. erythraea cells. These putative DEBS 2 and DEBS 3 proteins were purified and subjected to N-terminal sequence analysis. The protein sequences were entirely consistent with the and DEBS 3 proteins were purified and subjected to N-terminal sequence analysis. The protein sequences were entirely consistent with the translation start sites predicted from the DNA sequences of ORFs 2 and 3. A third high molecular weight protein co-purified with DEBS 2 and DEBS 3 and had an N-terminal sequence that matched a protein sequence translated from the DNA sequence some 155 base pairs upstream from the previously proposed start codon of ORF1.

Amino Acid Sequence↗