GENE CLONING, BIOCHEMICAL-CHARACTERIZATION AND PHYSIOLOGICAL-ROLE OF A THERMOSTABLE LOW-SPECIFICITY L-THREONINE ALDOLASE FROM ESCHERICHIA-COLI

The ItaE gene encoding for a thermostable low-specificity L-threonine aldolase, which catalyzes the cleavage of L-threonine/L-allo-threonine to glycine and acetaldehyde, was cloned from Escherichia coli GS245 b y the polymerase chain reaction. Construction and expression of the pl asmid pLTAE, which contained the ItaE gene under the control of the la c promoter, resulted in a 227-fold increase in the specific activity a bove the level detected in E. coli cells containing the control vector The enzyme is thermostable: it retained its full activity upon heatin g at 60 degrees C for 1 h. The enzyme was thus feasibly purified to ho mogeneity by heat treatment and butyl-Toyopearl column chromatography, and characterized. To reveal the physiological role of the enzyme, ge ne disruption was performed, Knockout of the ItaE gene of wild-type E. coli did not effect the cellular growth rate, while disruption of the ItaE gene of E. coli GS245, whose serine hydroxymethyltransferase gen e was knocked out, caused a significant decrease in the cellular growt h rate, suggesting that the threonine aldolase is not a major source o f cellular glycine in wild-type E. coli but catalyzes an alternative p athway for cellular glycine when serine hydroxymethyltransferase is in ert.