Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, X. Analysis of structural elements responsible for the differences in thermostability and activation by fructose 1,6-bisphosphate in the lactate dehydrogenases from B. stearothermophilus and B. caldolyticus by protein engineering.
The amino-acid sequences of the lactate dehydrogenases (LDH) from B. stearothermophilus and B. caldolyticus differ at only 10 positions. The properties of these enzymes however show substantial differences. The LDH from B. stearothermophilus is activated by Fru-P2 and has a higher thermostability (10 degrees C) than the enzyme from B. caldolyticus which cannot be activated by Fru-P2. To correlate these functional differences to the structural properties, we have constructed a set of hybrid- and point-mutants of the two LDHs. The amino acids at positions 207, 209B, and 209C could be identified to confer the property of activation by Fru-P2 to the enzymes. This part of the enzyme is to a large extent also responsible for the different thermostabilities of these two proteins.
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule, Zürich.
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D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D004789:Enzyme Activation; D004795:Enzyme Stability; D005635:Fructosediphosphates / Q000494:pharmacology; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D013696:Temperature
D005635:Fructosediphosphates; D007770:L-Lactate Dehydrogenase; C029063:fructose-1,6-diphosphate