Single amino acid substitutions can further increase the stability of a thermophilic L-lactate dehydrogenase |
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Authors: | Kallwass Helmut KW; Surewicz Witold K; Parris Wendy; Macfarlane Emma LA; Luyten Marcel A; Kay Cyril M; Gold Marvin; Jones JBryan |
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Affiliation: | 1Department of Chemistry, University of Toronto Toronto, Ontario, M5S 1A1
2Institute for Biological Sciences, National Research Council of Canada Ottawa, Ontario
3Department of Molecular and Medical Genetics, University of Toronto Toronto, Ontario, M5S 1A8
4MRC Group in Protein Structure and Function, Department of Biochemistry, University of Alberta Edmonton, Alberta, T6G 2H7, Canada |
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Abstract: | Lactate dehydrogenases are of considerable interest as stereospecificcatalysts in the chemical preparation of enantiomerically pure -hydroxyacid synthons. For such applications in synthetic organicchemistry it would be desirable to have enzymes which tolerateelevated temperatures for prolonged reaction times, to increaseproductivity and to extend then applicability to poor substrates.Here, two examples are reported of significant thermostabilizations,induced by sitedirected mutagenesis, of an already thermostableprotein, the L-lactate dehydrogenase (EC 1.1.1.27
EC]
, 35 kDa permonomer subunit) from Bacillus stearothermophilus. Thermal inactivationof this enzyme is accompanied by irreversible unfolding of thenative protein structure. The replacement of Argl71 by Tyr stabilizesthe enzyme against thermal inactivation and unfolding. Thisstabilizing effect appears to be based on improved interactionsbetween the subunits in the core of the active dimeric or tetramericforms of the enzyme. The thermal stability of L-lactate dehydrogenasevariants with an active site Arg residue, either in the 171(wild-type) or in the 102 position, is further increased bysulfate ions. The two stabilizing effects are additive, as foundfor the Argl71Tyr/ Gln1O2Arg double mutant, for which the stabilityof the protein in 100 mM sulfate solution reaches that of L-lactatedehydrogenases from extreme thermophiles. All mutant proteinsretain significant catalytic activity, both in the presenceand absence of stnhilfoing salts, and are viable catalysts inpreparative scale reactions. |
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Keywords: | active site mutations/ lactate dehydrogenase/ protein stabilization/ sulfate binding/ thermostable enzymes |
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