Organisation of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of genes flanking the polyketide synthase.

Analysis of the gene cluster from Streptomyces hygroscopicus that governs the biosynthesis of the polyketide immuno-suppressant rapamycin (Rp) has revealed that it contains three exceptionally large open reading frames (ORFs) encoding the modular polyketide synthase (PKS). Between two of these lies a fourth gene (rapP) encoding a pipecolate-incorporating enzyme that probably also catalyzes closure of the macrolide ring. On either side of these very large genes are ranged a total of 22 further ORFs before the limits of the cluster are reached, as judged by the identification of genes clearly encoding unrelated activities. Several of these ORFs appear to encode enzymes that would be required for Rp biosynthesis. These include two cytochrome P-450 monooxygenases (P450s), designated RapJ and RapN, an associated ferredoxin (Fd) RapO, and three potential SAM-dependent O-methyltransferases (MTases), RapI, RapM and RapQ. All of these are likely to be involved in 'late' modification of the macrocycle. The cluster also contains a novel gene (rapL) whose product is proposed to catalyze the formation of the Rp precursor, L-pipecolate, through the cyclodeamination of L-lysine. Adjacent genes have putative roles in Rp regulation and export. The codon usage of the PKS biosynthetic genes is markedly different from that of the flanking genes of the cluster.

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.

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.

The biosynthetic gene cluster for the polyketide immunosuppressant rapamycin.

The macrocyclic polyketides rapamycin and FK506 are potent immunosuppressants that prevent T-cell proliferation through specific binding to intracellular protein receptors (immunophilins). The cloning and specific alteration of the biosynthetic genes for these polyketides might allow the biosynthesis of clinically valuable analogues. We report here that three clustered polyketide synthase genes responsible for rapamycin biosynthesis in Streptomyces hygroscopicus together encode 14 homologous sets of enzyme activities (modules), each catalyzing a specific round of chain elongation. An adjacent gene encodes a pipecolate-incorporating enzyme, which completes the macrocycle. The total of 70 constituent active sites makes this the most complex multienzyme system identified so far. The DNA region sequenced (107.3 kbp) contains 24 additional open reading frames, some of which code for proteins governing other key steps in rapamycin biosynthesis.

6-Deoxyerythronolide-B synthase 2 from Saccharopolyspora erythraea. Cloning of the structural gene, sequence analysis and inferred domain structure of the multifunctional enzyme.

Sequencing of the eryA region of the erythromycin biosynthetic gene cluster from Saccharopolyspora erythraea has revealed another structural gene (ORF B), in addition to the previously characterised ORF A, which appears to encode a component of 6-deoxyerythronolide-B synthase, the enzyme that catalyses the first stage in the biosynthesis of the polyketide antibiotic erythromycin A. The nucleotide sequence of ORF B, which lies immediately adjacent to ORF A, has been determined. The predicted gene product of ORF B is a polypeptide of 374417 Da (3568 amino acids), which is highly similar to the product of ORF A and which likewise contains a number of separate domains, each with substantial amino acid sequence similarity to components of known fatty-acid synthases and polyketide synthases. The order of the predicted active sites along the chain from the N-terminus is 3-oxoacyl-synthase--acyltransferase--acyl-carrier-protein-- 3-oxoacyl-synthase--acyltransferase--dehydratase--enoylreductase-- oxoreductase--acyl-carrier-protein. The position of the dehydratase active site has been pinpointed for the first time for any polyketide synthase or vertebrate fatty-acid synthase. The predicted domain structure of 6-deoxyerythronolide-B synthase is strikingly similar to that previously established for vertebrate fatty-acid synthases. This analysis of the sequence supports the view that the erythromycin-producing polyketide synthase contains three multienzyme polypeptides, each of which accomplishes two successive cycles of polyketide chain extension. In this scheme, the role of the ORF B gene product is to accomplish extension cycles 3 and 4.

Cloning and sequence analysis of genes involved in erythromycin biosynthesis in Saccharopolyspora erythraea: sequence similarities between EryG and a family of S-adenosylmethionine-dependent methyltransferases.

The gene cluster (ery) responsible for production of the macrolide antibiotic erythromycin by Saccharopolyspora erythraea is also known to contain ermE, the gene conferring resistance to the antibiotic. The nucleotide sequence has been determined of a 4.5 kb portion of the biosynthetic gene cluster, from a region lying between 3.7 kb and 8.2 kb 3' of ermE. This has revealed the presence of four complete open reading frames, including the previously known ery gene eryG, which catalyses the last step in the biosynthetic pathway. Comparison of the amino acid sequence of EryG with the sequence of other S-adenosylmethionine (SAM)-dependent methyltransferases has revealed that one of the sequence motifs previously suggested to be part of the SAM-binding site is present not only in EryG but also in many other recently sequenced SAM-dependent methyltransferases. Previous genetic studies have shown that this region also contains gene(s) involved in hydroxylation of the intermediate 6-deoxyerythronolide B. One of the three other open reading frames (eryF) in fact shows very high sequence similarity to known cytochrome P450 hydroxylases. An adjacent gene (ORF5) shows a strikingly high degree of similarity to prokaryotic and eukaryotic acyltransferases and thioesterases.

An unusually large multifunctional polypeptide in the erythromycin-producing polyketide synthase of Saccharopolyspora erythraea.

Erythromycin A, a clinically important polyketide antibiotic, is produced by the Gram-positive bacterium Saccharopolyspora erythraea. In an arrangement that seems to be generally true of antibiotic biosynthetic genes in Streptomyces and related bacteria like S. erythraea, the ery genes encoding the biosynthetic pathway to erythromycin are clustered around the gene (ermE) that confers self-resistance on S. erythraea. The aglycone core of erythromycin A is derived from one propionyl-CoA and six methylmalonyl-CoA units, which are incorporated head-to-tail into the growing polyketide chain, in a process similar to that of fatty-acid biosynthesis, to generate a macrolide intermediate, 6-deoxyerythronolide B. 6-Deoxyerythronolide B is converted into erythromycin A through the action of specific hydroxylases, glycosyltransferases and a methyltransferase. We report here the analysis of about 10 kilobases of DNA from S. erythraea, cloned by chromosome 'walking' outwards from the erythromycin-resistance determinant ermE, and previously shown to be essential for erythromycin biosynthesis. Partial sequencing of this region indicates that it encodes the synthase. Our results confirm this, and reveal a novel organization of the erythromycin-producing polyketide synthase, which provides further insight into the mechanism of chain assembly.

Antibody-dependent cell-mediated cytotoxicity against IBR-infected bovine kidney cells by ruminant neutrophils: the role of lysosomal cationic protein.

Antibody-dependent cell-mediated cytotoxicity (ADCC) of infectious bovine rhinotracheitis (IBR)-infected bovine kidney cells (MDBK) by neutrophils was demonstrated. Neutrophils from bovine and sheep mammary exudate and peripheral blood, and also from human peripheral blood, were all active in the presence of anti-IBR antibody. The component of the ruminant neutrophil granules which was responsible for cytotoxicity appeared to be cationic protein since purified cationic protein lysed the virus-infected cells and heparin inhibited cytotoxicity. Human neutrophil cytotoxicity to herpes simplex virus (HSV)-infected human Chang liver cells was also inhibited by heparin. Human neutrophil cytotoxicity to IBR-infected bovine kidney cells did not appear to be mediated by cationic protein since it was inhibited by the chelators of oxidative intermediates DMSO, thiourea, tryptophane, benzoate and mannitol, and not by heparin.