Sequencing and expression of the gene encoding a cold-active citrate synthase from an Antarctic bacterium, strain DS2-3R.
The gene encoding citrate synthase from a novel bacterial isolate (DS2-3R) from Antarctica has been cloned, sequenced and over expressed in Escherichia coli. Both the recombinant enzyme and the native enzyme, purified from DS2-3R, are cold-active, with a temperature optimum of 31 degrees C. In addition the enzymes are rapidly inactivated at 45 degrees C, and show significant activity at 10 degrees C and below. Comparison of amino acid sequences indicates that DS2-3R citrate synthase is most closely related to the enzyme from gram-positive bacteria. The amino acid sequence of the DS2-3R enzyme shows several features previously recognised in other cold-active enzymes, including an extended surface loop, an increase in the occurrence of charged residues and a decrease in the number of proline residues in loops. Other changes observed in some psychrophilic enzymes, such as a decrease in isoleucine content and in arginine/(arginine+lysine) content, were not seen in this case.
Gerike
U
U
Centre for Extremophile Research, Department of Biology and Biochemistry, University of Bath, UK.
Danson
M J
MJ
Russell
N J
NJ
Hough
D W
DW
D000595:Amino Acid Sequence; D000864:Antarctic Regions; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D002950:Citrate (si)-Synthase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D003080:Cold Temperature; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006094:Gram-Positive Bacteria / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D002950:Citrate (si)-Synthase