Phosphoenolpyruvate carboxylase from Streptomyces coelicolor A3(2): purification of the enzyme, cloning of the ppc gene and over-expression of the protein in a streptomycete.
Phosphoenolpyruvate carboxylase [PEPC; orthophosphate:oxaloacetate carboxy-lyase (phosphorylating); EC 4.1.1.31] is a major anaplerotic enzyme in the polyketide producer Streptomyces coelicolor A3(2). PEPC was purified from S. coelicolor and the amino-acid sequences of four tryptic peptides were determined. Synthetic oligonucleotides based on the sequences of two of the peptides hybridized to the same bands in various restriction-enzyme digests of S. coelicolor genomic DNA. This hybridization allowed molecular cloning of an 8 kb BamHI fragment of genomic DNA. Partial DNA sequencing of this fragment showed that it could encode amino acid sequences similar to those of PEPC from other microorganisms. A BamHI/PstI fragment was subcloned into the streptomycete high-copy-number plasmid vector pIJ486 and transferred into Streptomyces lividans. The resulting strain over-expressed PEPC activity 21-fold and also over-expressed a protein with a subunit of 100,000 M(r), the same as that of purified S. coelicolor PEPC.
Bramwell
H
H
Department of Biochemistry, Glasgow University, Scotland, U.K.
Nimmo
H G
HG
Hunter
I S
IS
Coggins
J R
JR
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D010730:Phosphoenolpyruvate Carboxylase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D015183:Restriction Mapping; D013302:Streptomyces / Q000201:enzymology*
D004269:DNA, Bacterial; D010730:Phosphoenolpyruvate Carboxylase