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 triose phosphate isomerase for metabolizing dihydroxyacetone phosphate (DHAP) . In the present studies, the methylglyoxal synthase gene in Escherich ia coli has been cloned and sequenced. The identified open reading fra me (ORF) codes for a polypeptide of 152 amino acids, consistent with t he 17 kDa purified protein. The sequence of this protein is not simila r to any other protein of known function, including the functionally s imilar protein triosephosphate isomerase. The methylglyoxal synthase g ene was amplified by PCR, subcloned into the pET16B expression vector, and expressed in the host E, coli BL21(DE3). Sequence comparison of t he methylglyoxal protein and related ORFs from four different bacteria l species revealed that four aspartic acid and no glutamic acid residu es are absolutely conserved. The function of the four aspartic acid re sidues was tested by mutating them to either asparagine or glutamic ac id. Thermal denaturation, CD spectroscopy, and gel filtration experime nts showed that the mutant enzymes had the same secondary and quaterna ry structure as the wild-type enzyme. Kinetic characterization of both Asp 71 and Asp 101 mutant proteins shows reduced k(cat)/K-m 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 ar e involved with protecting the enzyme from DHAP or reactive intermedia tes along the catalytic pathway. In combination with the results of a- phosphoglycolate binding studies, a catalytic mechanism is proposed.