Biosynthesis of ansatrienin by Streptomyces collinus: Cell-free transformations of cyclohexene- and cyclohexadienecarboxylic acids.

A stable-isotope assay was used to analyze the effectiveness of various perdeuterated short-chain acyl coenzyme A (acyl-CoA) compounds as starter units for straight-and branched-chain fatty acid biosynthesis in cell extracts of Streptomyces collinus. In these extracts perdeuterated isobutyryl-CoA was converted to isopalmitate (a branched-chain fatty acid), while butyryl-CoA was converted to palmitate (a straight-chain fatty acid). These observations are consistent with previous in vivo analyses of fatty acid biosynthesis in S. collinus, which suggested that butyryl-CoA and isobutyryl-CoA function as starter units for palmitate and isopalmitate biosynthesis, respectively. Additionally, in vitro analysis demonstrated that acetyl-CoA can function as a starter unit for palmitate biosynthesis. Palmitate biosynthesis and isopalmitate biosynthesis in these cell extracts were both effectively inhibited by thiolactomycin, a known type II fatty acid synthase inhibitor. In vivo experiments demonstrated that concentrations of thiolactomycin ranging from 0.1 to 0.2 mg/ml produced both a dramatic decrease in the cellular levels of branched-chain fatty acids and a surprising three-to fivefold increase in the cellular levels of the straight-chain fatty acids palmitate and myristate. Additional in vivo incorporation studies with perdeuterated butyrate suggested that, in accord with the in vitro studies, the biosynthesis of the palmitate from butyryl-CoA decreases in the presence of thiolactomycin. In contrast, in vivo incorporation studies with perdeuterated acetate demonstrated that the biosynthesis of palmitate from acetylCoA increases in the presence of thiolactomycin. These observations clearly demonstrate that isobutyryl-CoA is a starter unit for isopalmitate biosynthesis and that either acetyl-CoA or butyryl-CoA can be a starter unit for palmitate biosynthesis in S. collinus. However, the pathway for palmitate biosynthesis from acetyl-CoA is less sensitive to thiolactomycin, and it is suggested that the basis for this difference is in the initiation step.

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“…Thiolactomycin was provided by Pfizer Incorporated. CoA thioesters were prepared by the mixed-anhydride method as previously described (26).…”

Section: Methodsmentioning

confidence: 99%

In vivo and in vitro effects of thiolactomycin on fatty acid biosynthesis in Streptomyces collinus

et al. 1997

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“…1) (13,42,43). It is thought that most of the steps in this process occur with the carboxylic acid activated as a coenzyme A thioester and that the final step in the pathway is the conversion of 1-cyclohexenylcarbonyl coenzyme A (CoA) (compound 3) to cyclohexylcarbonyl CoA (compound 4) (42,43,(48)(49)(50). An enzyme, with the designation 1-cyclohexenylcarbonyl CoA reductase (ChcA), capable of catalyzing this ␣,␤-double bond reduction has been purified and characterized from S. collinus (51).…”

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confidence: 99%

“…ChcA was assayed by spectrophotometric determination of NADPH oxidation at 340 nm in buffer C (consisting of 50 mM potassium phosphate and 10% [wt/vol] glycerol at pH 7.3) as previously described (50,51). Coenzyme A thioesters were prepared by using ethylchlorofor- mate as previously described (50,51). trans-4,5-Dihydroxycyclohex-1-enecarboxylic acid, trans-3,4-dihydroxycyclohexa-1,5-dienecarboxylic acid, and 2-cyclohexenecarboxylic acid were prepared as described previously (42,49).…”

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confidence: 99%

Cloning and characterization of the gene encoding 1-cyclohexenylcarbonyl coenzyme A reductase from Streptomyces collinus

et al. 1996

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We report the cloning of the gene encoding the 1-cyclohexenylcarbonyl coenzyme A reductase (ChcA) of Streptomyces collinus, an enzyme putatively involved in the final reduction step in the formation of the cyclohexyl moiety of ansatrienin from shikimic acid. The cloned gene, with a proposed designation of chcA, encodes an 843-bp open reading frame which predicts a primary translation product of 280 amino acids and a calculated molecular mass of 29.7 kDa. Highly significant sequence similiarity extending along almost the entire length of the protein was observed with members of the short-chain alcohol dehydrogenase superfamily. The S. collinus chcA gene was overexpressed in Escherichia coli by using a bacteriophage T7 transient expression system, and a protein with a specific ChcA activity was detected. The E. coli-produced ChcA protein was purified and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as the enoyl-coenzyme A reductase protein prepared from S. collinus. The enzyme demonstrated the ability to catalyze, in vitro, three of the reductive steps involved in the formation of cyclohexanecarboxylic acid. An S. collinus chcA mutant, constructed by deletion of a genomic region comprising the 5 end of chcA, lost the ChcA activity and the ability to synthesize either cyclohexanecarboxylic acid or ansatrienin. These results suggest that chcA encodes the ChcA that is involved in catalyzing multiple reductive steps in the pathway that provides the cyclohexanecarboxylic acid from shikimic acid.

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“…2-Cyclohexenecarboxylic acid, 1(6)-cyclohexadienecarboxylic acid, and 1,3-cyclohexadienecarboxylic acid were synthesized according to previously described procedures (25).…”

Section: Methodsmentioning

confidence: 99%

“…More recently, a conversion of 1-cyclohexenecarboxylic acid, activated as its coenzyme A (CoA) thioester, to the corresponding thioester of cyclohexanecarboxylic acid in a partially purified cell-free system from S. collinus was observed (24). Transformations of various cyclohexene-and cyclohexadienecarboxylic acids by a cell extract of S. collinus have also been conducted (25). The conclusion from these studies, in accordance with in vivo studies (16a), was that the last three steps of the pathway were a reduction of the a,1 double bond of 1(6),2-cyclohexadienylcarbonyl-CoA (Fig.…”

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confidence: 99%

Purification and characterization of a novel enoyl coenzyme A reductase from Streptomyces collinus

et al. 1992

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A novel NADPH-dependent enoyl reductase, catalyzing the conversion of 1-cyclohexenylcarbonyl coenzyme A (1-cyclohexenylcarbonyl-CoA) to cyclohexylcarbonyl-CoA, was purified to homogeneity from Streptomyces coUinus. This enzyme, a dimer with subunits of identical Mr (36,000), exhibits a Km of 1.5 0.3 ,uM for NADPH and 25 + 3 ,uM for 1-cyclohexenylcarbonyl-CoA. It has a pH optimum of 7.5, is most active at 30°C, and is inhibited by both divalent cations and thiol reagents. Two internal peptide sequences were obtained.

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Biosynthesis of ansatrienin by Streptomyces collinus: Cell-free transformations of cyclohexene- and cyclohexadienecarboxylic acids.

Cited by 15 publications

References 18 publications

In vivo and in vitro effects of thiolactomycin on fatty acid biosynthesis in Streptomyces collinus

In vivo and in vitro effects of thiolactomycin on fatty acid biosynthesis in Streptomyces collinus

Cloning and characterization of the gene encoding 1-cyclohexenylcarbonyl coenzyme A reductase from Streptomyces collinus

Purification and characterization of a novel enoyl coenzyme A reductase from Streptomyces collinus

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