Cloning, isolation and characterization of the Thermotoga maritima KDPG aldolase

Abstract

The Thermotoga maritima aldolase gene has been cloned into a T7 expression vector and overexpressed in Escherichia coli. The preparation yields 470 UL −1 of enzyme at a specific activity of 9.4 U mg −1. During retroaldol cleavage of KDPG, the enzyme shows a k cat that decreases with decreasing temperature. A more than offsetting decrease in K m yields an enzyme that is more efficient at 40 °C than at 70 °C. The substrate specificity of the enzyme was evaluated in the synthetic direction with a range of aldehyde substrates. Although the protein shows considerable structural homology to KDPG aldolases from mesophilic sources, significant differences in substrate specificity exist. A preparative scale reaction between 2-pyridine carboxaldehyde and pyruvate provided product of the same absolute configuration as mesophilic enzymes, but with diminished stereoselectivity.