Cloning, isolation and characterization of the Thermotoga maritima KDPG aldolase.
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 degrees C than at 70 degrees 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.
Griffiths
Jennifer S
JS
Department of Chemistry, Duke University, Durham, NC 27708, USA.
Wymer
Nathan J
NJ
Njolito
Eugenia
E
Niranjanakumari
S
S
Fierke
Carol A
CA
Toone
Eric J
EJ
D000446:Aldehyde-Lyases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005942:Gluconates / Q000378:metabolism; D007700:Kinetics; D013379:Substrate Specificity; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology*
D005942:Gluconates; C081217:2-keto-3-deoxy-6-phosphogluconate; D000446:Aldehyde-Lyases; C100131:phospho-2-keto-3-deoxy-gluconate aldolase