Gene cloning, biochemical characterization and physiological role of a thermostable low-specificity L-threonine aldolase from Escherichia coli.
The ltaE 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 by the polymerase chain reaction. Construction and expression of the plasmid pLTAE, which contained the ltaE gene under the control of the lac promoter, resulted in a 227-fold increase in the specific activity above the level detected in E. coli cells containing the control vector. The enzyme is thermostable: it retained its full activity upon heating at 60 degrees C for 1 h. The enzyme was thus feasibly purified to homogeneity by heat treatment and butyl-Toyopearl column chromatography, and characterized. To reveal the physiological role of the enzyme, gene disruption was performed. Knockout of the ltaE gene of wild-type E. coli did not effect the cellular growth rate, while disruption of the ltaE gene of E. coli GS245, whose serine hydroxymethyltransferase gene was knocked out, caused a significant decrease in the cellular growth rate, suggesting that the threonine aldolase is not a major source of cellular glycine in wild-type E. coli but catalyzes an alternative pathway for cellular glycine when serine hydroxymethyltransferase is inert.