The gene encoding glyoxalase I from Pseudomonas putida: cloning, overexpression, and sequence comparisons with human glyoxalase I.
The gene encoding glyoxalase I (GlxI) from Pseudomonas putida has been cloned into the high-expression plasmid pBTacI. In the presence of IPTG, JM109 cells transformed with this vector give expression levels of GlxI 4000-fold higher than wild-type Escherichia coli. Contrary to a previous report, the nucleotide sequence of the gene encodes a 173-amino-acid polypeptide. Edman analysis indicates that the predicted N-terminal methionine is lost post-translationally to yield a 19407-Da protein. Mass spectrometry of the intact protein, and of the peptides generated from treatment with CNBr, does not indicate any additional post-translational modifications of the enzyme. Contrary to previous conclusions, there are no major regions of dissimilarity between the human and bacterial enzymes.
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Department of Chemistry, University of Maryland Baltimore County 21228.
Creighton
D J
DJ
Antoine
M
M
Fenselau
C
C
Lovett
P S
PS
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli; D005798:Genes, Bacterial; D006801:Humans; D007791:Lactoylglutathione Lyase / Q000235:genetics*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010957:Plasmids; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D007791:Lactoylglutathione Lyase