- Structural characterization of an L-fuculose-1-phosphate aldolase from Klebsiella pneumoniae
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Fuculose phosphate aldolases play an important role in glycolysis and gluconeogenesis pathways. L-fuculose 1-phosphate aldolase catalyzes the reversible cleavage of L-fuculose 1-phosphate to DHAP and L-lactaldehyde. Class II aldolases found in bacteria are linked to pathogenesis of human pathogens, and have potential applications in the biosynthesis of carbohydrates and other chiral compounds. Here we report the structure of a putative L-fuculose 1-phosphate aldolase (KpFucA) from the nosocomial pathogen Klebsiella pneumoniae to 1.85 ? resolution. The enzyme crystallizes in space group P422 with one monomer per asymmetric unit. Analytical ultracentrifugation analysis confirms that KpFucA is a tetramer in solution. A magnesium ion cofactor and sulfate ion were identified in the active pocket. Enzyme activity assays confirmed that KpFcuA has a strong preference for L-fuculose 1-phosphate as a substrate, but can also catalyze the cleavage of fructose-1,6-bisphosphate and glucose-6-phosphate. This work should provide a starting point for further investigation of the role of KpFucA in K. pneumoniae pathogenesis or in industrial applications.
- Lou, Xiaorui,Zhang, Jianyu,Liu, Shimeng,Wang, Runhao,Li, Weiping,Liu, Ruihua,Zhang, Qionglin,Bartlam, Mark
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- Enzymatic vs. Fermentative Synthesis: Thermostable Glucose Dehydrogenase Catalyzed Regeneration of NAD(P)H for use in Enzymatic Synthesis
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Procedures are described for regeneration of reduced nicotinamide cofactors NADH and NADPH from NAD(P) based on glucose and a thermostable glucose dehydrogenase from Bacillus cereus immobilized in polyacrylamide gels.The turnover number for NAD in a synthesis of 200 mmol of D-lactate is 40000.Application of this system to other syntheses is demonstrated with preparations of ethyl (R)-4-chloro-3-hydroxybutanoate, (R)-2,2,2-trifluoro-1-phenylethanol, ethyl (S)-3-hydroxyvalerate, (S)-lactaldehyde dimethyl acetal, and (S)-3-hydroxybutanal dimethyl acetal.Further investigation of the kinetics regarding the thermoresistance of glucose dehydrogenase in the presence of NaCl has been carried out, and it appears that the enhancement by NaCl of the thermal stability of the enzyme is approximately third order.The immobilized glucose dehydrogenase incubated at 55 deg C, pH 7.5, for 7 days is still fully active while many other enzymes are completely inactivated in 1-2 days.Addition of NaCl enhances the thermal stability more significantly than the immobilization does, and a remarkable increase in thermal stability was observed with these two combined factors.The half-life of the immobilized glucose dehydrogenase at 55 deg C in a buffer (pH 7.0-7.5) containing 1 M NaCl is more than 30 days compared to 3 min for the free enzyme, corresponding to an overall ca. 50000-fold increase in thermal stability.
- Wong, Chi-Huey,Drueckhammer, Dale G.,Sweers, Henri M.
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p. 4028 - 4031
(2007/10/02)
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