Identification of catalytic bases in the active site of Escherichia coli methylglyoxal synthase: cloning, expression, and functional characterization of conserved aspartic acid residues.
Methylglyoxal synthase provides bacteria with an alternative to triosephosphate isomerase for metabolizing dihydroxyacetone phosphate (DHAP). In the present studies, the methylglyoxal synthase gene in Escherichia coli has been cloned and sequenced. The identified open reading frame (ORF) codes for a polypeptide of 152 amino acids, consistent with the 17 kDa purified protein. The sequence of this protein is not similar to any other protein of known function, including the functionally similar protein triosephosphate isomerase. The methylglyoxal synthase gene was amplified by PCR, subcloned into the pET16B expression vector, and expressed in the host E. coli BL21(DE3). Sequence comparison of the methylglyoxal protein and related ORFs from four different bacterial species revealed that four aspartic acid and no glutamic acid residues are absolutely conserved. The function of the four aspartic acid residues was tested by mutating them to either asparagine or glutamic acid. Thermal denaturation, CD spectroscopy, and gel filtration experiments showed that the mutant enzymes had the same secondary and quaternary structure as the wild-type enzyme. Kinetic characterization of both Asp 71 and Asp 101 mutant proteins shows reduced kcat/Km by 10(3)- and 10(4)-fold respectively, suggesting that they are both intimately involved in catalysis. A time-dependent inhibition of both Asp 20 and Asp 91 asparagine mutants by DHAP suggests that these two residues are involved with protecting the enzyme from DHAP or reactive intermediates along the catalytic pathway. In combination with the results of 2-phosphoglycolate binding studies, a catalytic mechanism is proposed.
Saadat
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Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226, USA.
Harrison
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D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D019757:Carbon-Oxygen Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D002384:Catalysis; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000302:isolation & purification; D004795:Enzyme Stability; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D006016:Glycolates / Q000494:pharmacology; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000187:drug effects; D010710:Phosphates; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D017421:Sequence Analysis
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006016:Glycolates; D010710:Phosphates; D011994:Recombinant Proteins; D001224:Aspartic Acid; D019757:Carbon-Oxygen Lyases; C021578:methylglyoxal synthase; C013193:phosphoglycolate