Cloning, overexpression and mechanistic studies of carboxyphosphonoenolpyruvate mutase from Streptomyces hygroscopicus.
The enzyme carboxyphosphonoenolpyruvate mutase catalyses the formation of one of the two C-P bonds in bialaphos, a potent herbicide isolated from Streptomyces hygroscopicus. The gene encoding the enzyme has been cloned from a subgenomic library from S. hygroscopicus by colony hybridisation using an exact nucleotide probe. An open reading frame has been identified that encodes a protein of molecular mass 32700 Da, in good agreement with the subunit molecular mass of the carboxyphosphonoenolpyruvate mutase recently isolated from this source [Hidaka, T., Imai, S., Hara, O., Anzai, H., Murakami, T., Nagaoka, K. & Seto, H. (1990) J. Bacteriol. 172, 3066-3072]. The gene shares significant sequence similarity with that of phosphoenolpyruvate mutase, an enzyme that catalyses the related interconversion of phosphoenolpyruvate and phosphonopyruvate. When the carboxyphosphonoenolpyruvate-mutase gene was subcloned into the vector pET11a, the mutase was expressed as about 20% of the total soluble cellular protein in Escherichia coli. The mutase has been purified to homogeneity in three steps in 40% yield. With malate dehydrogenase/NADH, (hydroxyphosphinyl)pyruvate gives (hydroxyphosphinyl)lactate (kcat 164 s-1 and Km 680 microM) and this spectrophotometric assay for the product of the mutase reaction has been employed in the mechanistic studies. The kinetics for the mutase reaction have been evaluated for the substrate, carboxyphosphonoenolpyruvate, and for the putative reaction intermediate carboxyphosphinopyruvate, both of which have been prepared by chemical synthesis. Carboxyphosphonoenolpyruvate is converted to (hydroxyphosphinyl)pyruvate with a kcat of 0.020 s-1 and a Km of 270 microM, and carboxyphosphinopyruvate is converted to (hydroxyphosphinyl)pyruvate with a kcat of 7.6 x 10(-4) s-1 and a Km of 2.2 microM. Although the exogenously added intermediate is not kinetically competent, these results suggest that the mechanism for the mutase reaction involves an initial rearrangement to the intermediate carboxyphosphinopyruvate, followed by decarboxylation to yield the product (hydroxyphosphinyl)pyruvate.
Pollack
S J
SJ
Department of Chemistry, Harvard University, Cambridge, Massachusetts.
Freeman
S
S
Pompliano
D L
DL
Knowles
J R
JR
D000595:Amino Acid Sequence; D001483:Base Sequence; D002845:Chromatography; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D015698:Genomic Library; D007700:Kinetics; D007773:Lactates; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D016366:Open Reading Frames; D009943:Organophosphorus Compounds / Q000138:chemical synthesis / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism; D010770:Phosphotransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017875:Phosphotransferases (Phosphomutases); D011773:Pyruvates; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D004269:DNA, Bacterial; D007773:Lactates; D009838:Oligodeoxyribonucleotides; D009943:Organophosphorus Compounds; D011773:Pyruvates; D011994:Recombinant Proteins; C077742:carboxyphosphoenolpyruvic acid; C077744:(hydroxyphosphinyl)pyruvic acid; C077746:carboxyphosphinopyruvic acid; D010728:Phosphoenolpyruvate; C077745:(hydroxyphosphinyl)lactic acid; D010770:Phosphotransferases; D017875:Phosphotransferases (Phosphomutases); C064363:carboxyphosphonoenolpyruvate phosphonomutase