Cloning and expression of the Zymomonas mobilis pyruvate kinase gene in Escherichia coli.
The homotetrameric pyruvate kinases (PK) constitute a fine example of allosteric enzymes subjected to sophisticated regulatory mechanisms. We have cloned and sequenced the Zymomonas mobilis structural gene for the first prokaryotic dimeric PK, as an initial step toward understanding the peculiar properties of this enzyme. The deduced amino acid sequence of the pyk gene consists of 475 residues with a calculated molecular mass of 51.4kDa and exhibits up to 50% sequence identity with other PKs. Heterologous expression in Escherichia coli was not obtained from the native promoter, but only when the pyk gene was under the control of a strong inducible promoter when a ribosome-binding site was present upstream of the putative TTG start codon of the pyk gene. Kinetic characterization of PK in concentrated crude cell extracts showed that the enzyme is not activated by sugar phosphates or AMP but is slightly inhibited by ATP. Thus, PK of Z. mobilis is unique among the characterized prokaryotic PKs due to its high activity in the absence of any allosteric activator. Amino acid sequence alignments revealed that glutamate 381 may play a role in ineffective binding of the usual PK activator, fructose-1,6-bisphosphate.
Steiner
P
P
Institute of Biotechnology, ETH Zürich, CH-8093, Zürich, Switzerland.
Fussenegger
M
M
Bailey
J E
JE
Sauer
U
U
D020121:5' Untranslated Regions / Q000235:genetics; D000494:Allosteric Regulation; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D011770:Pyruvate Kinase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017355:Zymomonas / Q000201:enzymology / Q000235:genetics*
D020121:5' Untranslated Regions; D011770:Pyruvate Kinase