Biomedical subjects

P F Leadlay

Publications and source records attributed to P F Leadlay.

At least 19 recordsLinked to original sources

PubMed2002

Analysis of a C-methyltransferase gene (aviG1) involved in avilamycin biosynthesis in Streptomyces viridochromogenes Tü57 and complementation of a Saccharopolyspora erythraea eryBIII mutant by aviG1.

Streptomyces viridochromogenes Tü57 is the principal producer of avilamycin A. aviG1, a putative methyltransferase gene, was detected in the avilamycin biosynthetic gene cluster. To determine the function of aviG1, a targeted gene inactivation experiment was performed. The resulting chromosomal mutant, carrying an in-frame deletion in aviG1, was deficient in avilamycin production. aviG1 was used to complement an eryBIII mutant of the erythromycin A producer Saccharopolyspora erythraea [Gaisser, S., Bohm, G. A., Doumith, M., Raynal, M. C., Dhillon, N., Cortes, J. & Leadlay, P. F. (1998). Mol Gen Genet 258, 78-88]. The presence of erythromycin A in the culture supernatant of the complemented mutant indicated that L-mycarose biosynthesis could be restored and that AviG1 could take over the function of the C-methyltransferase EryBIII.

Amino Acid Sequence↗

PubMed2001

Role of type II thioesterases: evidence for removal of short acyl chains produced by aberrant decarboxylation of chain extender units.

BACKGROUND: Modular polyketide synthases (PKSs) function as molecular assembly lines in which polyketide chains are assembled by successive addition of chain extension units. At the end of the assembly line, there is usually a covalently linked type I thioesterase domain (TE I), which is responsible for release of the completed acyl chain from its covalent link to the synthase. Additionally, some PKS clusters contain a second thioesterase gene (TE II) for which there is no established role. Disruption of the TE II genes from several PKS clusters has shown that the TE II plays an important role in maintaining normal levels of antibiotic production. It has been suggested that the TE II fulfils this role by removing aberrant intermediates that might otherwise block the PKS complex. RESULTS: We show that recombinant tylosin TE II behaves in vitro as a TE towards a variety of N-acetylcysteamine and p-nitrophenyl esters. The trends of hydrolytic activity determined by the kinetic parameter k(cat)/K(M) for the analogues tested indicates that simple fatty acyl chains are effective substrates. Analogues that modelled aberrant forms of putative tylosin biosynthetic intermediates were hydrolysed at low rates. CONCLUSIONS: The behaviour of tylosin TE II in vitro is consistent with its proposed role as an editing enzyme. Aberrant decarboxylation of a malonate-derived moiety attached to an acyl carrier protein (ACP) domain may generate an acetate, propionate or butyrate residue on the ACP thiol. Our results suggest that removal of such groups is a significant role of TE II.

Carboxylic Acids↗

PubMed2001

Molecular basis of Celmer's rules: role of the ketosynthase domain in epimerisation and demonstration that ketoreductase domains can have altered product specificity with unnatural substrates.

BACKGROUND: Polyketides are structurally diverse natural products with a range of medically useful activities. Non-aromatic bacterial polyketides are synthesised on modular polyketide synthase multienzymes (PKSs) in which each cycle of chain extension requires a different 'module' of enzymatic activities. Attempts to design and construct modular PKSs that synthesise specified novel polyketides provide a particularly stringent test of our understanding of PKS structure and function. RESULTS: We show that the ketoreductase (KR) domains of modules 5 and 6 of the erythromycin PKS, housed in the multienzyme subunit DEBS3, exert an unexpectedly low level of stereochemical control in reducing the keto group of a synthetic analogue of the diketide intermediate. This led us to construct a hybrid triketide synthase based on DEBS3 with ketosynthase domain ketosynthase (KS)5 replaced by the loading module and KS1. The construct in vivo produced two major triketide stereoisomers, one expected and one surprising. The latter was of opposite configuration at three out of the four chiral centres: the branching alkyl centre was that produced by KS1 and, surprisingly, both hydroxyl centres produced by the reduction steps carried out by KR5 and KR6 respectively. CONCLUSIONS: These results demonstrate that the epimerising activity associated with module 1 of the erythromycin PKS can be conferred on module 5 merely by transfer of the KS1 domain. Moreover, the normally precise stereochemical control observed in modular PKSs is lost when KR5 and KR6 are challenged by an unfamiliar substrate, which is much smaller than their natural substrates. This observation demonstrates that the stereochemistry of ketoreduction is not necessarily invariant for a given KR domain and underlines the need for mechanistic understanding in designing genetically engineered PKSs to produce novel products.

Amino Acid Sequence↗

PubMed2001

Engineering a polyketide with a longer chain by insertion of an extra module into the erythromycin-producing polyketide synthase.

BACKGROUND: Modular polyketide synthases catalyse the biosynthesis of medically useful natural products by stepwise chain assembly, with each module of enzyme activities catalysing a separate cycle of polyketide chain extension. Domain swapping between polyketide synthases leads to hybrid multienzymes that yield novel polyketides in a more or less predictable way. No experiments have so far been reported which attempt to enlarge a polyketide synthase by interpolating additional modules. RESULTS: We describe here the construction of tetraketide synthases in which an entire extension module from the rapamycin-producing polyketide synthase is covalently spliced between the first two extension modules of the erythromycin-producing polyketide synthase (DEBS). The extended polyketide synthases thus formed are found to catalyse the synthesis of specific tetraketide products containing an appropriate extra ketide unit. Co-expression in Saccharopolyspora erythraea of the extended DEBS multienzyme with multienzymes DEBS 2 and DEBS 3 leads to the formation, as expected, of novel octaketide macrolactones. In each case the predicted products are accompanied by significant amounts of unextended products, corresponding to those of the unaltered DEBS PKS. We refer to this newly observed phenomenon as 'skipping'. CONCLUSIONS: The strategy exemplified here shows far-reaching possibilities for combinatorial engineering of polyketide natural products, as well as revealing the ability of modular polyketide synthases to 'skip' extension modules. The results also provide additional insight into the three-dimensional arrangement of modules within these giant synthases.

Cyclohexanones↗

PubMed2001

Chain initiation on the soraphen-producing modular polyketide synthase from Sorangium cellulosum.

BACKGROUND: Polyketides are structurally diverse natural products with a wide range of useful activities. Bacterial modular polyketide synthases (PKSs) catalyse the production of non-aromatic polyketides using a different set of enzymes for each successive cycle of chain extension. The choice of starter unit is governed by the substrate specificity of a distinct loading module. The unusual loading module of the soraphen modular PKS, from the myxobacterium Sorangium cellulosum, specifies a benzoic acid starter unit. Attempts to design functional hybrid PKSs using this loading module provide a stringent test of our understanding of PKS structure and function, since the order of the domains in the loading and first extension module is non-canonical in the soraphen PKS, and the producing strain is not an actinomycete. RESULTS: We have constructed bimodular PKSs based on DEBS1-TE, a derivative of the erythromycin PKS that contains only extension modules 1 and 2 and a thioesterase (TE) domain, by substituting one or more domains from the soraphen PKS. A hybrid PKS containing the soraphen acyltransferase domain AT1b instead of extension acyltransferase domain AT1 produced triketide lactones lacking a methyl group at C-4, as expected if AT1b catalyses the addition of malonyl-CoA during the first extension cycle on the soraphen PKS. Substitution of the DEBS1-TE loading module AT domain by the soraphen AT1a domain led to the production of 5-phenyl-substituted triketide lactone, as well as the normal products of DEBS1-TE. This 5-phenyl triketide lactone was also the product of a hybrid PKS containing the entire soraphen PKS loading module as well as part of its first extension module. Phenyl-substituted lactone was only produced when measures were simultaneously taken to increase the intracellular supply of benzoyl-CoA in the host strain of Saccharopolyspora erythraea. CONCLUSIONS: These results demonstrate that the ability to recruit a benzoate starter unit can be conferred on a modular PKS by the transfer either of a single AT domain, or of multiple domains to produce a chimaeric first extension module, from the soraphen PKS. However, benzoyl-CoA needs to be provided within the cell as a specific precursor. The data also support the respective roles previously assigned to the adjacent AT domains of the soraphen loading/first extension module. Construction of such hybrid actinomycete-myxobacterial enzymes should significantly extend the synthetic repertoire of modular PKSs.

Amino Acid Sequence↗

PubMed2001

Engineering of complex polyketide biosynthesis--insights from sequencing of the monensin biosynthetic gene cluster.

The biosynthesis of complex reduced polyketides is catalysed in actinomycetes by large multifunctional enzymes, the modular Type I polyketide synthases (PKSs). Most of our current knowledge of such systems stems from the study of a restricted number of macrolide-synthesising enzymes. The sequencing of the genes for the biosynthesis of monensin A, a typical polyether ionophore polyketide, provided the first genetic evidence for the mechanism of oxidative cyclisation through which polyethers such as monensin are formed from the uncyclised products of the PKS. Two intriguing genes associated with the monensin PKS cluster code for proteins, which show strong homology with enzymes that trigger double bond migrations in steroid biosynthesis by generation of an extended enolate of an unsaturated ketone residue. A similar mechanism operating at the stage of an enoyl ester intermediate during chain extension on a PKS could allow isomerisation of an E double bond to the Z isomer. This process, together with epoxidations and cyclisations, form the basis of a revised proposal for monensin formation. The monensin PKS has also provided fresh insight into general features of catalysis by modular PKSs, in particular into the mechanism of chain initiation.

Biotechnology↗

PubMed2001

New erythromycin derivatives from Saccharopolyspora erythraea using sugar O-methyltransferases from the spinosyn biosynthetic gene cluster.

Using a previously developed expression system based on the erythromycin-producing strain of Saccharopolyspora erythraea, O-methyltransferases from the spinosyn biosynthetic gene cluster of Saccharopolyspora spinosa have been shown to modify a rhamnosyl sugar attached to a 14-membered polyketide macrolactone. The spnI, spnK and spnH methyltransferase genes were expressed individually in the S. erythraea mutant SGT2, which is blocked both in endogenous macrolide biosynthesis and in ery glycosyltransferases eryBV and eryCIII. Exogenous 3-O-rhamnosyl-erythronolide B was efficiently converted into 3-O-(2'-O-methylrhamnosyl)-erythronolide B by the S. erythraea SGT2 (spnI) strain only. When 3-O-(2'-O-methylrhamnosyl)-erythronolide B was, in turn, fed to a culture of S. erythraea SGT2 (spnK), 3-O-(2',3'-bis-O-methylrhamnosyl)-erythronolide B was identified in the culture supernatant, whereas S. erythraea SGT2 (spnH) was without effect. These results confirm the identity of the 2'- and 3'-O-methyltransferases, and the specific sequence in which they act, and they demonstrate that these methyltransferases may be used to methylate rhamnose units in other polyketide natural products with the same specificity as in the spinosyn pathway. In contrast, 3-O-(2',3'-bis-O-methylrhamnosyl)-erythronolide B was found not to be a substrate for the 4'-O-methyltransferase SpnH. Although rhamnosylerythromycins did not serve directly as substrates for the spinosyn methyltransferases, methylrhamnosyl-erythromycins were obtained by subsequent conversion of the corresponding methylrhamnosyl-erythronolide precursors using the S. erythraea strain SGT2 housing EryCIII, the desosaminyltransferase of the erythromycin pathway. 3-O-(2'-O-methylrhamnosyl)-erythromycin D was tested and found to be significantly active against a strain of erythromycin-sensitive Bacillus subtilis.

Anti-Bacterial Agents↗

PubMed2000

Protection of radical intermediates at the active site of adenosylcobalamin-dependent methylmalonyl-CoA mutase.

Adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the interconversion of methylmalonyl-CoA and succinyl-CoA via radical intermediates generated by substrate-induced homolysis of the coenzyme carbon-cobalt bond. From the structure of methylmalonyl-CoA mutase it is evident that the deeply buried active site is completely shielded from solvent with only a few polar contacts made between the protein and the substrate. Site-directed mutants of amino acid His244, a residue close to the inferred site of radical chemistry, were engineered to investigate its role in catalysis. Two mutants, His244Ala and His244Gln, were characterized using kinetic and spectroscopic techniques. These results confirmed that His244 is not an essential residue. However, compared with that of the wild type, k(cat) was lowered by 10(2)- and 10(3)-fold for the His244Gln and His244Ala mutants, respectively, while the K(m) for succinyl-CoA was essentially unchanged in both cases. The primary kinetic tritium isotope effect (k(H)/k(T)) for the His244Gln mutant was 1.5 +/- 0.3, and tritium partitioning was now found to be dependent on the substrate used to initiate the reaction, indicating that the rearrangement of the substrate radical to the product radical was extremely slow. The His244Ala mutant underwent inactivation under aerobic conditions at a rate between 1 and 10% of the initial rate of turnover. The crystal structure of the His244Ala mutant, determined at 2.6 A resolution, indicated that the mutant enzyme is unaltered except for a cavity in the active site which is occupied by an ordered water molecule. Molecular oxygen reaching this cavity may lead directly to inactivation. These results indicate that His244 assists directly in the unusual carbon skeleton rearrangement and that alterations in this residue substantially lower the protection of reactive radical intermediates during catalysis.

Aerobiosis↗

PubMed2000

Novel octaketide macrolides related to 6-deoxyerythronolide B provide evidence for iterative operation of the erythromycin polyketide synthase.

BACKGROUND: The macrolide antibiotic erythromycin A, like other complex aliphatic polyketides, is synthesised by a bacterial modular polyketide synthase (PKS). Such PKSs, in contrast to other fatty acid and polyketide synthases which work iteratively, contain a separate set or module of enzyme activities for each successive cycle of polyketide chain extension, and the number and type of modules together determine the structure of the polyketide product. Thus, the six extension modules of the erythromycin PKS (DEBS) together catalyse the production of the specific heptaketide 6-deoxyerythronolide B. RESULTS: A mutant strain of the erythromycin producer Saccharopolyspora erythraea, which accumulates the aglycone intermediate erythronolide B, was found unexpectedly to produce two novel octaketides, both 16-membered macrolides. These compounds were detectable in fermentation broths of wild-type S. erythraea, but not in a strain from which the DEBS genes had been specifically deleted. From their structures, both of these octaketides appear to be aberrant products of DEBS in which module 4 has 'stuttered', that is, has catalysed two successive cycles of chain extension. CONCLUSIONS: The isolation of novel DEBS-derived octaketides provides the first evidence that an extension module in a modular PKS has the potential to catalyse iterative rounds of chain elongation like other type I FAS and PKS systems. The factors governing the extent of such 'stuttering' remain to be determined.

Anti-Bacterial Agents↗

PubMed2000

A defined system for hybrid macrolide biosynthesis in Saccharopolyspora erythraea.

The biological activity of polyketide antibiotics is often strongly dependent on the presence and type of deoxysugar residues attached to the aglycone core. A system is described here, based on the erythromycin-producing strain of Saccharopolyspora erythraea, for detection of hybrid glycoside formation, and this system has been used to demonstrate that an amino sugar characteristic of 14-membered macrolides (D-desosamine) can be efficiently attached to a 16-membered aglycone substrate. First, the S. erythraea mutant strain DM was created by deletion of both eryBV and eryCIII genes encoding the respective ery glycosyltransferase genes. The glycosyltransferase OleG2 from Streptomyces antibioticus, which transfers L-oleandrose, has recently been shown to transfer rhamnose to the oxygen at C-3 of erythronolide B and 6-deoxyerythronolide B. In full accordance with this finding, when oleG2 was expressed in S. erythraea DM, 3-O-rhamnosyl-erythronolide B and 3-O-rhamnosyl-6-deoxyerythronolide B were produced. Having thus validated the expression system, endogenous aglycone production was prevented by deletion of the polyketide synthase (eryA) genes from S. erythraea DM, creating the triple mutant SGT2. To examine the ability of the mycaminosyltransferase TylM2 from Streptomyces fradiae to utilise a different amino sugar, tylM2 was integrated into S. erythraea SGT2, and the resulting strain was fed with the 16-membered aglycone tylactone, the normal TylM2 substrate. A new hybrid glycoside was isolated in good yield and characterized as 5-O-desosaminyl-tylactone, indicating that TylM2 may be a useful glycosyltransferase for combinatorial biosynthesis. 5-O-glucosyl-tylactone was also obtained, showing that endogenous activated sugars and glycosyltransferases compete for aglycone in these cells.

Anti-Bacterial Agents↗

PubMed2000

Efficient purification and kinetic characterization of a bimodular derivative of the erythromycin polyketide synthase.

Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), are giant multienzymes that biosynthesize a number of clinically important natural products. The modular nature of PKSs suggests the possibility of a combinatorial approach to the synthesis of novel bioactive polyketides, but the efficacy of such a strategy depends critically on gaining fundamental insight into PKS structure and function, most directly through experiments with purified PKS proteins. Several recent investigations into important aspects of the activity of these enzymes have used only partially purified proteins (often 3-4% of total protein), reflecting how difficult it is to purify these multienzymes in amounts adequate for kinetic and structural analysis. We report here the steady-state kinetic analysis of a typical bimodular PKS, 6-deoxyerythronolide B synthase 1-thioesterase (DEBS 1-TE), purified from recombinant Saccharopolyspora erythraea JCB101 by a new, high-yielding procedure consisting of three steps: ammonium sulfate precipitation, hydrophobic interaction chromatography and size-exclusion chromatography. The method provides 13-fold purification with a recovery of 11% of the applied PKS activity. The essentially homogeneous synthase exhibits an intrinsic methylmalonyl-CoA hydrolase activity, which competes with polyketide chain extension. The most reliable value for the kcat for synthesis of (3S,5R)-dihydroxy-(2R,4R)-dimethyl-n-heptanoic acid-delta-lactone is 0.84 min-1, and the apparent Km for (2RS)-methylmalonyl-CoA is 17 microM. This kcat is approximately 10-fold lower than the value reported previously for a differently engineered version of the truncated PKS, DEBS 1+TE. The difference likely reflects the fact that the DEBS 1-TE contains a hybrid acyl carrier protein (ACP) domain in its second module, which lowers its catalytic efficiency.

Acyl Coenzyme A↗

PubMed1999

A chain initiation factor common to both modular and aromatic polyketide synthases.

Antibiotic-producing polyketide synthases (PKSs) are enzymes responsible for the biosynthesis in Streptomyces and related filamentous bacteria of a remarkably broad range of bioactive metabolites, including antitumour aromatic compounds such as mithramycin and macrolide antibiotics such as erythromycin. The molecular basis for the selection of the starter unit on aromatic PKSs is unknown. Here we show that a component of aromatic PKS, previously named 'chain-length factor', is a factor required for polyketide chain initiation and that this factor has decarboxylase activity towards malonyl-ACP (acyl carrier protein). We have re-examined the mechanism of initiation on modular PKSs and have identified as a specific initiation factor a domain of previously unknown function named KSQ, which operates like chain-length factor. Both KSQ and chain-length factor are similar to the ketosynthase domains that catalyse polyketide chain extension in modular multifunctional PKSs and in aromatic PKSs, respectively, except that the ketosynthase domain active-site cysteine residue is replaced by a highly conserved glutamine in KSQ and in chain-length factor. The glutamine residue is important both for decarboxylase activity and for polyketide synthesis.

Acyl Carrier Protein↗

PubMed1999

Structural elucidation studies of erythromycins by electrospray tandem mass spectrometry.

Erythromycin A (EryA) was studied by electrospray ionisation tandem mass spectrometry (ESI-MS/MS) with the aim of developing a methodology for the structural elucidation of novel erythromycins developed by biological synthetic methods. Skimmer dissociation along with sequential mass spectrometry studies (up to MS5) have been employed in this study. In the low-resolution MS/MS analysis of the polyketides, there are several fragment ions that are easily assigned to various neutral losses. These have all been confirmed by accurate-mass measurements. There is also a series of peaks due to ring opening and fragmentation that can only be assigned by high-resolution MSn analysis. Further experiments were performed in deuterated media (D2O/CD3OD 50%) which, along with the high-resolution MSn of erythromycin analogues, has enabled us to identify some of the steps in the ring fragmentation, particularly the loss of the polyketide starter acid. This is an essential step for determining structural alterations in the novel polyketides, but further labelling experiments and studies on more erythromycin analogues are required before the complete fragmentation pathway can be confirmed.

Anti-Bacterial Agents↗

PubMed1999

Structural elucidation studies of erythromycins by electrospray tandem mass spectrometry II.

Erythromycin A (EryA), sec-butyl erythromycin B (SEryB), oleandomycin (Olean) and a synthetic derivative, roxithromycin (Rox), were used to investigate the fragmentation of polyketide macrolide antibiotics by collision induced dissociation (CID) tandem mass spectrometry (MS/MS). Analyses were performed with two commercially available mass spectrometers: a Q-TOF hybrid quadrupole time-of-flight instrument and a BioApex II (4.7 Tesla) Fourier transform ion cyclotron resonance (FTICR) instrument both equipped with electrospray ionisation (ESI) sources. One of the first fragmentation processes is the loss of an H(2)O molecule from the [M+H](+) ion. EryA has three hydroxyl groups on the polyketide ring and loses three H(2)O molecules during CID. This study indicates that these facts are not necessarily related. Deuterium exchange experiments were carried out in order to isotopically label free hydroxyl groups. (18)O-exchange experiments were also carried out in order to label the carbonyl group at the 9-position. In EryA and its analogue the first H(2)O loss shifts in mass from loss of 18 Da to loss of 20 Da in deuterated solvents. For both molecules the loss also shifts in mass from loss of 18 Da to loss of 20 Da during the (18)O-exchange experiments. This suggests that the first loss of H(2)O is from the 9-position carbonyl group, indicating that this, and not the nitrogen of the amino sugar, is the site of protonation of the activated MH(+) ions. For Rox the initial loss of H(2)O is replaced by loss of the 9-position oxime group, the rest of the fragmentation sequence being the same as for EryA. For Olean, there is no H(2)O loss from the parent ion. The results have allowed the proposal of a mechanism for the first loss of H(2)O in the EryA MS/MS fragmentation.

Anti-Bacterial Agents↗

PubMed1999

Knowledge-based design of bimodular and trimodular polyketide synthases based on domain and module swaps: a route to simple statin analogues.

BACKGROUND: Polyketides are structurally diverse natural products that have a range of medically useful activities. Nonaromatic bacterial polyketides are synthesised on modular polyketide synthase (PKS) multienzymes, in which each cycle of chain extension requires a different 'module' of enzymatic activities. Attempts to design and construct modular PKSs that synthesise specified novel polyketides provide a particularly stringent test of our understanding of PKS structure and function. RESULTS: We have constructed bimodular and trimodular PKSs based on DEBS1-TE, a derivative of the erythromycin PKS that contains only modules 1 and 2 and a thioesterase (TE), by substituting multiple domains with appropriate counterparts derived from the rapamycin PKS. Hybrid PKSs were obtained that synthesised the predicted target triketide lactones, which are simple analogues of cholesterol-lowering statins. In constructing intermodular fusions, whether between modules in the same or in different proteins, it was found advantageous to preserve intact the acyl carrier protein-ketosynthase (ACP-KS) didomain that spans the junction between successive modules. CONCLUSIONS: Relatively simple considerations govern the construction of functional hybrid PKSs. Fusion sites should be chosen either in the surface-accessible linker regions between enzymatic domains, as previously revealed, or just inside the conserved margins of domains. The interaction of an ACP domain with the adjacent KS domain, whether on the same polyketide or not, is of particular importance, both through conservation of appropriate protein-protein interactions, and through optimising molecular recognition of the altered polyketide chain in the key transfer of the acyl chain from the ACP of one module to the KS of the downstream module.

Amino Acid Sequence↗

PubMed1999

Molecular basis of Celmer's rules: the role of two ketoreductase domains in the control of chirality by the erythromycin modular polyketide synthase.

BACKGROUND: Polyketides are compounds that possess medically significant activities. The modular nature of the polyketide synthase (PKS) multienzymes has generated interest in bioengineering new PKSs. Rational design of novel PKSs, however, requires a greater understanding of the stereocontrol mechanisms that operate in natural PKS modules. RESULTS: The N-acetyl cysteamine (NAC) thioester derivative of the natural beta-keto diketide intermediate was incubated with DEBS1-TE, a derivative of the erythromycin PKS that contains only modules 1 and 2. The reduction products of the two ketoreductase (KR) domains of DEBS1-TE were a mixture of the (2S, 3R) and (2R,3S) isomers of the corresponding beta-hydroxy diketide NAC thioesters. Repeating the incubation using a DEBS1-TE mutant that only contains KR1 produced only the (2S,3R) isomer. CONCLUSIONS: In contrast with earlier results, KR1 selects only the (2S) isomer and reduces it stereospecifically to the (2S, 3R)-3-hydroxy-2-methyl acyl product. The KR domain of module 1 controls the stereochemical outcome at both methyl-and hydroxyl-bearing chiral centres in the hydroxy diketide intermediate. Earlier work showed that the normal enzyme-bound ketoester generated in module 2 is not epimerised, however. The stereochemistry at C-2 is therefore established by a condensation reaction that exclusively gives the (2R)-ketoester, and the stereo-chemistry at C-3 by reduction of the keto group. Two different mechanisms of stereochemical control, therefore, operate in modules 1 and 2 of the erythromycin PKS. These results should provide a more rational basis for designing hybrid PKSs to generate altered stereochemistry in polyketide products.

Catalysis↗

PubMed1998

Stabilization of radical intermediates by an active-site tyrosine residue in methylmalonyl-CoA mutase.

The adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the reversible rearrangement of methylmalonyl-CoA into succinyl-CoA by a free-radical mechanism. The recently solved X-ray crystal structure of methylmalonyl-CoA mutase from Propionibacterium shermanii has shown that tyrosine 89 is an active-site residue involved in substrate binding. The role of tyrosine 89, a conserved residue among methylmalonyl-CoA mutases, has been investigated by using site-directed mutagenesis to replace this residue with phenylalanine. The crystal structure of the Tyr89Phe mutant was determined to 2.2 A resolution and was found to be essentially superimposable on that of wild-type. Mutant and wild-type enzyme have very similar KM values, but kcat for the Tyr89Phe mutant is 580-fold lower than for wild-type. The rate of release of tritium from 5'-[3H]adenosylcobalamin during the enzymatic reaction and its rate of appearance in substrate and product were measured. The tritium released was found to partition unequally between methylmalonyl-CoA and succinyl-CoA, in a ratio of 40:60 when the reaction was initiated by addition of methylmalonyl-CoA and in a ratio of 10:90 when the reaction was initiated by addition of succinyl-CoA. The overall release of tritium was four times faster when succinyl-CoA was used as substrate. The tritium isotope effect on the enzyme catalyzed hydrogen transfer, measured with methylmalonyl-CoA as a substrate, was kH/kT = 30, which is within the expected range for a full primary kinetic tritium isotope effect. The different partitioning of tritium, dependent upon which substrate was used, and the normal value for the kinetic tritium isotope effect contrast markedly with the behavior of wild-type mutase. It appears that the loss of a single interaction involving the hydroxyl group of tyrosine 89 both affects the stability of radical intermediates and decreases the rate of interconversion of the substrate- and product-derived radicals.

Amino Acid Substitution↗

PubMed1998

Construction of new vectors for high-level expression in actinomycetes.

A new integrative vector (pCJR24) was constructed for use in the erythromycin producer Saccharopolyspora erythraea and in other actinomycetes. It includes the pathway-specific activator gene actII-ORF4 from the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor. The actI promoter and the associated ribosome binding site are located upstream of an NdeI site (5'-CATATG-3') which encompasses the actI start codon allowing protein(s) to be produced at high levels in response to nutritional signals if these signals are faithfully mediated by the ActII-ORF4 activator. Several polyketide synthase genes were cloned in pCJR24 and overexpressed in S. erythraea after integration of the vector into the chromosome by homologous recombination, indicating the possibility that the S. coelicolor promoter/activator functions appropriately in S. erythraea. pCJR24-mediated recombination was also used to place the entire gene set for the erythromycin-producing polyketide synthase under the control of the actI promoter. The resulting strain produced copious quantities of erythromycins and precursor macrolides when compared with wild-type S. erythraea. The use of this system provides the means for rational strain improvement of antibiotic-producing actinomycetes.

Actinomycetales↗