T R Dafforn — Page 2 | PubMed Health

PubMed1997

Protein engineering tests of a homology model of Plasmodium falciparum lactate dehydrogenase.

This paper describes the testing of a homology model of Plasmodium falciparum lactate dehydrogenase (pfLDH) by protein engineering. The model had been validated in structural terms. It suggests explanations of the unusual properties of pfLDH (compared with all other LDHs). These unusual features are a lack of substrate inhibition, high activity with the synthetic coenzyme 3-acetylpyridine adenine dinucleotide (APAD+) and changes in residues at previously conserved positions. pfLDH shows several amino acid insertions and deletions in an alignment with protein sequences from all other known LDHs. The most notable is a five amino acid insertion into the active-site loop. In addition, a conserved serine at position 163 is replaced by leucine. The results showed that when the unique pfLDH structural features were engineered into Bacillus stearothermophilus lactate dehydrogenase, the thermophilic enzyme acquired the properties previously uniquely associated with the malarial enzyme. We conclude that the homology model of the malarial enzyme is adequate for the prediction of successful redesigns and, in the regions tested, is accurate.

Amino Acid Sequence↗

PubMed1997

Correlation of the enzyme activities of Bacillus stearothermophilus lactate dehydrogenase on three substrates with the results of molecular dynamics/energy minimization conformational searching.

Current methods for reengineering enzyme substrate specificities rely heavily on the use of static x-ray crystallographic models. In this article we detail the use of a molecular mechanics approach for suggesting regions of Bacillus stearothermophilus L-lactate dehydrogenase (EC 1.1.1.27) involved in substrate specificity, and hence areas of interest for protein engineers. The approach combines molecular dynamics with energy minimization (MD/EM) to search the conformational space available to a 15-A sphere of the ternary complex centered on the catalytic histidine. The search is carried out by calculating a 30-ps dynamics trajectory at 300 K and minimizing structures at 1-ps intervals. The protocol has been performed on 14 systems containing different combinations of substrate and mutant/wt LDH. In order to discover which interactions are important in defining substrate specificity, eight conformational parameters representing substrate-active site interactions were measured in each of the 420 minimized structures. These parameters were then compared to the measured catalytic activity of the protein-substrate combinations. These comparisons show that arginine 109 orientation is a major determining factor in LDH specificity. Using this methodology it is possible to estimate the catalytic activity of proteins of varied sequence by computer simulation before synthesis.

Catalysis↗