Purification of Crotonyl‐CoA Reductase from <i>Streptomyces collinus</i> and Cloning, Sequencing and Expression of the Corresponding Gene in <i>Escherichia coli</i>

Wallace, Kimberlee K.; Bao, Zhuo‐Yao ‐Y; Dai, Hong; DiGate, Russell J.; Schuler, Gregory D.; Speedie, Marilyn K.; Reynolds, Kevin A.

doi:10.1111/j.1432-1033.1995.954_3.x

Cited by 57 publications

(67 citation statements)

References 59 publications

(45 reference statements)

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“…The ChcA, however, catalyzes an enoyl thioester reduction in contrast to the alcohol dehydrogenation catalyzed by other members of the SCAD superfamily (38). It is interesting that the ChcA is not related to a crotonyl CoA reductase recently isolated from S. collinus (62) or any other enoyl thioester reductases involved in either fatty acid biosynthesis (14,30) or secondary metabolism (19,20). The origin of other enzymes in the unusual pathway to cyclohexanecarboxylic acid might provide similar surprises and will be the subject of future investigations.…”

Section: Discussionmentioning

confidence: 99%

“…S. collinus was grown in a twostage fermentation as previously described (63) by using a liquid medium containing 0.05 g of magnesium sulfate heptahydrate, 0.1 g of ammonium sulfate, 0.01 g of calcium chloride dihydrate, 0.5 g of potassium monobasic, 0.05 g of sodium citrate, and 1 ml of a trace element solution (1.25 mg of magnesium chloride tetrahydrate, 0.28 mg of ferrous sulfate heptahydrate, and 0.48 mg of zinc sulfate in 125 ml of water) in 100 ml of water at pH 6.5. A gas chromatography-mass spectrometry fatty acid analysis of S. collinus cells harvested from a 48-h fermentation was carried out as previously described (62). When required, the liquid medium in the second fermentation stage was supplemented with cyclohexanecarboxylic acid (0.4 mM) at the time of inoculation.…”

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confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

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

et al. 1996

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“…However, no detectable sequence similarity exists between TER and crotonyl-CoA reductase. Furthermore, S. collinus crotonyl-CoA reductase shows no activity with NADH in contrast to Euglena TER (56,57). Crotonyl-CoA reductase provides butyryl-CoA units for synthesis of polyketids (58), but is again distinct from Euglena TER.…”

Section: Trans-2-enoyl-coa Reductasementioning

confidence: 99%

Mitochondrial trans-2-Enoyl-CoA Reductase of Wax Ester Fermentation from Euglena gracilis Defines a New Family of Enzymes Involved in Lipid Synthesis

Hoffmeister¹,

Piotrowski

Nowitzki³

et al. 2005

Journal of Biological Chemistry

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Under anaerobiosis, Euglena gracilis mitochondria perform a malonyl-CoA independent synthesis of fatty acids leading to accumulation of wax esters, which serve as the sink for electrons stemming from glycolytic ATP synthesis and pyruvate oxidation. An important enzyme of this unusual pathway is trans-2-enoyl-CoA reductase (EC 1.3.1.44), which catalyzes reduction of enoyl-CoA to acyl-CoA. Trans-2-enoyl-CoA reductase from Euglena was purified 1700-fold to electrophoretic homogeneity and was active with NADH and NADPH as the electron donor. The active enzyme is a monomer with molecular mass of 44 kDa. The amino acid sequence of tryptic peptides determined by electrospray ionization mass spectrometry were used to clone the corresponding cDNA, which encoded a polypeptide that, when expressed in Escherichia coli and purified by affinity chromatography, possessed trans-2-enoyl-CoA reductase activity close to that of the enzyme purified from Euglena. Trans-2-enoyl-CoA reductase activity is present in mitochondria and the mRNA is expressed under aerobic and anaerobic conditions. Using NADH, the recombinant enzyme accepted crotonyl-CoA (k m ‫؍‬ 68 M) and trans-2-hexenoyl-CoA (k m ‫؍‬ 91 M). In the crotonyl-CoA-dependent reaction, both NADH (k m ‫؍‬ 109 M) or NADPH (k m ‫؍‬ 119 M) were accepted, with 2-3-fold higher specific activities for NADH relative to NADPH. Trans-2-enoyl-CoA reductase homologues were not found among other eukaryotes, but are present as hypothetical reading frames of unknown function in sequenced genomes of many proteobacteria and a few Gram-positive eubacteria, where they occasionally occur next to genes involved in fatty acid and polyketide biosynthesis. Trans-2-enoyl-CoA reductase assigns a biochemical activity, NAD(P)H-dependent acyl-CoA synthesis from enoylCoA, to one member of this gene family of previously unknown function.

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“…Accordingly, these systems must have either an additional pathway for butyryl-CoA formation or the potential to initiate straight-chain fatty acid biosynthesis from a starter unit other than butyryl-CoA (38). Evidence for the former of these suggestions has been the isolation and characterization of a crotonyl-CoA reductase from S. collinus (37). This enzyme is thought to play a key role in catalyzing the last reductive step in a malonyl-CoA-independent biosynthesis of butyryl-CoA from two acetyl-CoA molecules (37).…”

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confidence: 99%

“…Evidence for the former of these suggestions has been the isolation and characterization of a crotonyl-CoA reductase from S. collinus (37). This enzyme is thought to play a key role in catalyzing the last reductive step in a malonyl-CoA-independent biosynthesis of butyryl-CoA from two acetyl-CoA molecules (37).…”

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

et al. 1997

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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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Purification of Crotonyl‐CoA Reductase from Streptomyces collinus and Cloning, Sequencing and Expression of the Corresponding Gene in Escherichia coli

Cited by 57 publications

References 59 publications

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

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

Mitochondrial trans-2-Enoyl-CoA Reductase of Wax Ester Fermentation from Euglena gracilis Defines a New Family of Enzymes Involved in Lipid Synthesis

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

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