Appendix. Cloning and sequence of the gene encoding enzyme E-1 from the methionine salvage pathway of Klebsiella oxytoca

• Published in: The Journal of biological chemistry Vol. 268; no. 33; pp. 24792 - 24795
• Main Authors: Balakrishnan, R, Frohlich, M, Rahaim, P T, Backman, K, Yocum, R R
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 25.11.1993; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Base Sequence; Biological and medical sciences; Cloning, Molecular; DNA, Bacterial; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Genes, Bacterial; Klebsiella - enzymology; Methionine - metabolism; Miscellaneous; Molecular Sequence Data; Phosphopyruvate Hydratase - genetics; Phosphopyruvate Hydratase - isolation & purification; Phosphopyruvate Hydratase - metabolism; Phosphoric Monoester Hydrolases - genetics; Phosphoric Monoester Hydrolases - isolation & purification; Phosphoric Monoester Hydrolases - metabolism; Enzyme; Phosphoprotein phosphatase; Methionine; Phosphoric monoester hydrolases; Primary structure; Esterases; Lyases; Biosynthesis; Klebsiella oxytoca; Aminoacid; Bacteria; Hydrolases; Molecular cloning; Carbon-oxygen lyases; Phosphopyruvate hydratase; Enterobacteriaceae; Hydro-lyases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)74534-7

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.