OVEREXPRESSION AND MUTAGENESIS OF THE CATALYTIC DOMAIN OF DIHYDROLIPOAMIDE ACETYLTRANSFERASE FROM SACCHAROMYCES-CEREVISIAE

The inner core domain (residues ~221-454) of the dihydrolipoamide acetyltransferase component (E2p) of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae has been overexpressed in Escherichia coli strain JM105 via the expression vector pKK233-2. The truncated E2p was purified to apparent homogeneity. It exhibited catalytic activity (acetyl transfer from [1-14C]acetyl-CoA to dihydrolipoamide) very similar to that of wild-type E2p. The appearance of the truncated and wild-type E2p was also very similar, as observed by negative-stain electron microscopy, namely, a pentagonal dodecahedron. These findings demonstrate that the active site of E2p from S. cerevisiae resides in the inner core domain, i.e., catalytic domain, and that this domain alone can undergo self-assembly. The purified truncated E2p showed a tendency to aggregate. Aggregation was prevented by genetically engineered attachment of the interdomain linker segment (residues ~ 181-220) to the catalytic domain. All dihydrolipoamide acyl-transferases contain the sequence His-Xaa-Xaa-Xaa-Asp-Gly near their carboxyl termini. By analogy with chloramphenicol acetyltransferase, the highly conserved His and Asp residues were postulated to be involved in the catalytic mechanism [Guest, J. R. (1987) FEMS Microbiol. Lett. 44, 417-422]. Substitution of the sole His residue in the S. cerevisiae truncated E2p, His-427, by Asn or Ala by site-directed mutagenesis did not have a significant effect on the kcat or Km values of the truncated E2p. However, the Asp-431 → Asn, Ala, or Glu substitutions resulted in a 16-, 24-, and 3.7-fold reduction, respectively, in kcat, with little change in Km values. These findings indicate that a His residue is not involved in the catalytic mechanism of E2p from S. cerevisiae but that Asp-431 plays an important role. Whether this role is structural or catalytic remains to be established.© 1990, American Chemical Society. All rights reserved.