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.
Hewitt
C O
CO
Molecular Recognition Centre, University of Bristol School of Medical Sciences, UK.
Sessions
R B
RB
Dafforn
T R
TR
Holbrook
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D015195:Drug Design; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics* / Q000493:pharmacokinetics / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D015202:Protein Engineering; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D009243:NAD; C005941:3-acetylpyridine adenine dinucleotide; D007770:L-Lactate Dehydrogenase