Appendix. Cloning and sequence of the gene encoding enzyme E-1 from the methionine salvage pathway of Klebsiella oxytoca.
The methionine salvage pathway converts the methylthioribose moiety of 5'-(methylthio)-adenosine to methionine via a series of biochemical steps. One enzyme active in this pathway, a bifunctional enolase-phosphatase called E-1 that promotes oxidative cleavage of the synthetic substrate 2,3-diketo-1-phosphohexane to 2-keto-pentanoate, has been purified from Klebsiella pneumoniae and is characterized in the preceding paper (Myers, R., Wray, J., Fish, S., and Abeles, R. H. (1993) J. Biol. Chem. 268, 24785-24791). We synthesized degenerate oligonucleotides corresponding to portions of the amino terminus of E-1. These oligonucleotides were used as polymerase chain reaction primers on whole genomic DNA from Klebsiella oxytoca. This resulted in an 82-base pair DNA fragment that was used as a hybridization probe to obtain a clone of the E-1 gene from a K. oxytoca gene library. The DNA sequence of the E-1 coding region was determined, and the amino acid sequence of E-1 was deduced. E-1 appears to represent a novel class of enzymes since no homology to known enzymes was found. Cloning the gene from K. oxytoca on a multicopy plasmid leads to overproduction of E-1 enzyme that has properties indistinguishable from those of the enzyme from K. pneumoniae.
Balakrishnan
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OmniGene, Inc., Cambridge, Massachusetts 02139-9002.
Frohlich
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Rahaim
P T
PT
Backman
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K
Yocum
R R
RR
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D005798:Genes, Bacterial; D007709:Klebsiella / Q000201:enzymology*; D008715:Methionine / Q000378:metabolism*; D008969:Molecular Sequence Data; D010751:Phosphopyruvate Hydratase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
D004269:DNA, Bacterial; D008715:Methionine; D010744:Phosphoric Monoester Hydrolases; D010751:Phosphopyruvate Hydratase