Overexpression and mutagenesis of the catalytic domain of dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae

Abstract

The inner core domain (residues approximately 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 approximately 181-220) to the catalytic domain. All dihydrolipoamide acyltransferases 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Saccharomyces cerevisiae Atf1p is an alcohol acetyltransferase and a thioesterase in vitro

“…This suggests that the conserved active‐site histidine is not absolutely required for substrate hydrolysis; we did not assess ester production by H191A, and it may be that the alcohol acyltransferase reaction is abolished in this mutant. The H191A mutation leaves K M essentially unchanged, and this was anticipated since results with other acyltransferases suggest that the catalytic histidine can play only a minor role in determining substrate binding affinity (Lewendon et al, ; Suzuki et al, ; Niu et al, ; Hsiao et al, ). The exception to this general trend is at C14 chain lengths, where the K M of H191A is ~20 times larger than that of wild‐type protein.…”

Atomic Structure of the Cubic Core of the Pyruvate Dehydrogenase Multienzyme Eomplex

“…In both enzymes (Fig. This interpretation is supported by the mutagenesis studies on the E. coli enzyme (27) but not by those on the yeast molecule (28), where the replacement of this His by Ala does not affect the activity. Such a conformation allows a hydrogen bond between the imidazole nitrogen NM1 and the carbonyl 0 of the same amino acid.…”

Structure and Function of the Catalytic Domain of the Dihydrolipoyl Acetyltransferase Component in Escherichia coli Pyruvate Dehydrogenase Complex

“…Direct Demonstration of Acetyl Transfer between E2p DomainsIt had been reported that E2p and independently expressed E2pCD catalyze the model reaction using acetyl-CoA as acetyl donor and a DL-dihydrolipoamide as acetyl acceptor (DL-dihydrolipoamide replacing the dihydrolipoyl moieties covalently bound to the lipoyl domain) in the reaction reversed from the physiological reaction (see Reaction 1) (23,24,(45)(46)(47). The acetyltransferase activity had been measured in a coupled assay with phosphotransacetylase, where the formation of a thioester bond (S-acetyldihydrolipoamide) was monitored at 240 nm (233 nm) (23,24,46), or by detection of the radioactive S-acetyldihydrolipoamide formed from [1-14 C]acetyl-CoA (45, 47).…”