Mevalonate catabolism in Pseudomonas: Cloning and sequencing of HMG -coenzyme A reductase
Abstract
Protein sequencing of HMG-CoA reductase from Pseudomonas sp. M gave 45% of the predicted structure from which two specific oligonucleotide probes were designed for use in cloning the gene. The probes were used to screen a restriction fragment-enriched Pseudomonas:pUC19 library. The positive clone obtained was then used as a probe for screening a Pseudomonas:EMBL4 library from which a 14 kb positive insert was obtained. A 2.1 kb PstI/HindIII fragment was sequenced and shown to encode HMG-CoA reductase based on comparison to the cyanogen bromide peptide amino acid composition and sequence data. The HMG-CoA reductase gene encodes a hydrophilic protein of MW 45,538. Comparison of Pseudomonas sp. M and mammalian HMG-CoA reductases gives two blocks of homology with approximately 33% homology over 29% of the Pseudomonas enzyme sequence, which may reflect their functional relatedness. Comparison of cysteine-containing peptides in Pseudomonas sp. M HMG-CoA reductase and other four-electron oxidoreductases shows some similarity with UDP-D-glucose dehydrogenase and mammalian HMG-CoA reductase. The 500 bp segment upstream from HMG-CoA reductase includes a putative ribosome binding site and a promoter that contains a array of direct- and inverted repeats. The putative promoter also contains sequences homologous to those found in Klebsiella and Pseudomonas promoters. An open reading frame begins immediately following the HMG-GoA reductase gene and extends beyond the fragment sequenced. The potential ribosome binding site for this open reading frame overlaps the end of the HMG-CoA reductase gene and therefore the putative protein may be translationally coupled to HMG-CoA reductase in a mevalonate catabolic operon. Analysis of the amino terminal segment of the putative protein showed that it contains a consensus sequence thought to be involved in metal binding.
Degree
Ph.D.
Advisors
Rodwell, Purdue University.
Subject Area
Biochemistry
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