Analysis of the nucleotide sequence of the Streptomyces glaucescens tcmI genes provides key information about the enzymology of polyketide antibiotic biosynthesis.

Bibb, Mervyn J.; Bíró, Sándor; Motamedi, Haideh; Collins, John; Hutchinson, C. Richard

doi:10.1002/j.1460-2075.1989.tb08414.x

Cited by 261 publications

(122 citation statements)

References 48 publications

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“…5), such as the emodin to chrysophanol conversion in secalonic acid biosynthesis [46], the versicolorin A to sterigmatocystin conversion in aflatoxin biosynthesis [47], the biosynthesis of the granaticin and actinorhodin antibiotics [48,49], and the biosynthesis of the tetranomycin anthracyclin [50].…”

Section: Discussionmentioning

confidence: 99%

Polyhydroxynaphthalene reductase involved in melanin biosynthesis in Magnaporthe grisea

Vidal-Cros

Viviani

Labesse

et al. 1994

European Journal of Biochemistry

115

102

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During the biosynthesis of fungal melanin, tetrahydroxynaphthalene reductase catalyzes the NADPH-dependent reduction of 1,3,6,S-tetrahydroxynaphthalene (T,HN) into (+)-scytalone and 1,3,8-trihydroxynaphthalene into (-)-vermelone. The enzyme from Magnaporthe grisea, the fungus responsible for rice blast disease, has been purified to homogeneity. It is a tetramer of four identical 30-kDa subunits. A full-length cDNA clone of about 1 kb encoding T4HN reductase has been isolated from a cDNA library constructed in the LZAP I1 vector and characterized. The clone contains a 846-bp open reading frame. Translation of the DNA sequence gave a 282-residue amino acid sequence with a calculated molecular mass of 29.9 kDa. Sequences corresponding to the aminoterminal part and three internal proteolytic peptides were present in the translated sequence. T4HN reductase exhibits characteristics of the short-chain alcohol dehydrogenase family. The reductase shares 56% identity with a putative ketoreductase involved in aflatoxin biosynthesis in Aspergillus parasiticus.Melanin, a high-molecular-mass black pigment, is synthesized by numerous pathogenic fungi [l, 21 such as Verticillium dahliae, Cochliobolus rniyabeanus and Magnaporthe grisea, the agent of rice blast disease. Mutants of those pathogens lacking the capability to synthesize melanin lose their ability to penetrate the host leaf and, by the way, their pathogenicity [3, 41. The melanin biosynthesis is thus a good target for the design of antifungal agents. The biosynthesis of melanin has been elucidated with mutants accumulating shunt products and exhibiting typical phenotypes (albino, orange) [5]. Fungal melanin is derived from a pentaketide intermediate that is cyclized into 1,3,6,8-tetrahydroxynaphthalene (T,HN) (Fig. 1). The last steps consist of a series of reductions and dehydrations, leading to 1,8-dihydroxynaphthalene via (+)scytalone, 2,3,8-trihydroxynaphthalene (T,HN) and (-)vermelone. Polymerization of 1 ,8-dihydroxynaphthalene yields melanin [6]. In contrast with the intermediates, the enzymes carrying out these transformations are much less characterized. Despite the cloning by complementation of a melanin biosynthetic gene of Colletotrickum lagenariuin [7] presumably involved in the pentaketide cyclization step and the recent isolation of a gene cluster involved in melanin biosynthesis in Alternaria alternata [S], neither the sequences nor the corresponding enzymes are available. Recent studies have focused on the dehydration step. The complete purification of scytalone dehydratase from C. miyabeanus [9], as well as preliminary crystallographic studies on the M . grisea enzyme [lo], has been reported. However, no reductase had been described until now, although inhibitors of this enzyme are already known and largely employed as fungicides, such as tricyclazole [ l l , 121. Experiments carried out with crude extracts from M . grisea have shown that a single enzyme performs the two reduction steps [13] and that this reductase is NADPH-dependent [14]. We prese...

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Section: Discussionmentioning

confidence: 99%

Polyhydroxynaphthalene reductase involved in melanin biosynthesis in Magnaporthe grisea

Vidal-Cros

Viviani

Labesse

et al. 1994

European Journal of Biochemistry

115

102

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“…The tcmK gene of S. glaucescens GLA.0 encodes a 45-kDa [~-keto acyl synthase that plays an early role in the synthesis of the polyketide antibiotic tetracenomycin C (ORF1 of Bibb et al, 1989;Gramajo et al, 1991). A 1.7-kb NdeI-tcmK-HindIII fragment, with the NdeI site overlapping the start codon of tcmK, was inserted between the NdeI and HindIII sites of pIJ6021 to give pIJ6024.…”

Section: (B) Application Of Plj6021mentioning

confidence: 99%

Construction of thiostrepton-inducible, high-copy-number expression vectors for use in Streptomyces spp.

Takano

White

Thompson

et al. 1995

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“…Recent evidence from the Steinbüchel laboratory has been interpreted to indicate that in vitro there are 25 polymer chains synthesized per molecule of C. Vinosum PHA synthase, which implies that there is a termination step (29). In animal fatty acid synthase and in some members of the polyketide synthase family, a thioesterase domain containing an essential serine is required for the hydrolysis step or lactone formation (30)(31)(32)(33). Thus, an alternative role for the conserved serine in PHA synthases might be controlling the chain length in a fashion that remains to be established.…”

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PHA Synthase from Chromatium vinosum: Cysteine 149 Is Involved in Covalent Catalysis

et al. 1998

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Polyhydroxyalkanoate synthase (PHA) from Chromatium Vinosum catalyzes the conversion of 3-hydroxybutyryl-CoA (HB-CoA) to polyhydroxybutyrate (PHB) and CoA. The synthase is composed of a ∼1:1 mixture of two subunits, PhaC and PhaE. Size-exclusion chromatography indicates that in solution PhaC and PhaE exist as large molecular weight aggregates. The holo-enzyme, PhaEC, has a specific activity of 150 units/mg. Each subunit was cloned, expressed, and purified as a (His) 6 -tagged construct. The PhaC-(His) 6 protein catalyzed polymerization with a specific activity of 0.9 unit/mg; the PhaE-(His) 6 protein was inactive (specific activity <0.001 unit/mg). Addition of PhaE-(His) 6 to PhaC-(His) 6 increased the activity several 100-fold. To investigate the priming step of the polymerization process, the PhaEC was incubated with a trimer of HB-CoA in which the terminal hydroxyl was replaced with tritium ( Sequencing by ion trap mass spectrometry showed that they were identical and that they each contained an altered cysteine (C149). One peptide contained the [ 3 H]-sT while the other two contained, in addition to the [ 3 H]-sT, one and two additional monomeric HBs, respectively. Mutation of C149 to alanine gave inactive synthase. The remaining two cysteines of PhaC, 292 and 130, were also mutated to alanine. The former had wild-type (wt) activity, while the latter had 0.004 wt % activity and was capable of making polymer. A mechanism is proposed in which PhaC contains all the elements essential for catalysis and the polymerization proceeds by covalent catalysis using C149 and potentially C130.Polyhydroxyalkanoates (PHAs 1 ) are polyoxoesters with properties that range from elastomers to thermoplastics (1-4). They are produced by a wide range of bacteria when they are placed in an environment of nutrient limitation (5). Copolymers of polyhydroxybutyrate (PHB) and polyhydroxyvalerate in the correct ratio have properties similar to the petrochemically based polypropylenes, the major component of bulk plastics (6). In 1997, the US market for thermoplastics was on the order of 40 million tons per year (7). PHAs have recently received much attention because they are biodegradable and can be generated from biorenewable sources: bacteria and plants (8). The major focus of many investigators is to make their production economically competitive with the polypropylenes. To achieve this goal, the requirements for the polymerization process need to be established. This paper focuses on the PHA synthase from Chromatium Vinosum which catalyzes the formation of PHB from 3-hydroxybutyryl-CoA (HB-CoA). Evidence is presented that two cysteines and covalent catalysis play an important role in the initiation and elongation of the polymerization process.PHA synthases from 20 organisms have now been identified. They have been divided into three classes based on their substrate specificity and subunit composition (9). The class I synthases, with the Ralstonia eutropha synthase as a prototype, are composed of a single polypeptide (∼65 k...

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Analysis of the nucleotide sequence of the Streptomyces glaucescens tcmI genes provides key information about the enzymology of polyketide antibiotic biosynthesis.

Cited by 261 publications

References 48 publications

Polyhydroxynaphthalene reductase involved in melanin biosynthesis in Magnaporthe grisea

Polyhydroxynaphthalene reductase involved in melanin biosynthesis in Magnaporthe grisea

Construction of thiostrepton-inducible, high-copy-number expression vectors for use in Streptomyces spp.

PHA Synthase from Chromatium vinosum: Cysteine 149 Is Involved in Covalent Catalysis

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