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 s teps. One enzyme active in this pathway, a bifunctional enolase-phosph atase called E-1 that promotes oxidative cleavage of the synthetic sub strate 2,3-diketo-1-phosphohexane to 2-ketopentanoate, has been purifi ed from Klebsiella pneumoniae and is characterized in the preceding pa per (Myers, R., Wray, J., Fish, S., and Abeles, R. H. (1993) J. Biol. Chem. 268, 24785-24791). We synthesized degenerate oligonucleotides co rresponding to portions of the amino terminus of E-1. These oligonucle otides were used as polymerase chain reaction primers on whole genomic DNA from Klebsiella oxytoca. This resulted in an 82-base pair DNA fra gment 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 c oding region was determined, and the amino acid sequence of E-1 was de duced. E-1 appears to represent a novel class of enzymes since no homo logy to known enzymes was found. Cloning the gene from K. oxytoca on a multicopy plasmid leads to overproduction of E-1 enzyme that has prop erties indistinguishable from those of the enzyme from K. pneumoniae.