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 kcat that decreases with decreasing temperature. A more than offsetting decrease in Km 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. © 2002 Elsevier Science Ltd. All rights reserved.