Abstract
Shikimate 5-dehydrogenase (SKDH; EC 1.1.1.25) catalyzes the reversible reduction of 3-dehydroshikimate to shikimate and is a key enzyme in the aromatic amino acid biosynthesis pathway. The shikimate 5-dehydrogenase gene, aroE, from Archaeoglobus fulgidus was cloned and overexpressed in Escherichia coli. The recombinant enzyme purified as a homodimer and yielded a maximum specific activity of 732 U/mg at 87 degrees C (with NADP+ as coenzyme). Apparent Km values for shikimate, NADP+, and NAD+ were estimated at 0.17+/-0.03 mM, 0.19+/-0.01 mM, and 11.4+/-0.4 mM, respectively. The half-life of the A. fulgidus SKDH is 2 h at the assay temperature (87 degrees C) and 17 days at 60 degrees C. Addition of 1 M NaCl or KCl stabilized the enzyme's half-life to approximately 70 h at 87 degrees C and approximately 50 days at 60 degrees C. This work presents the first kinetic analysis of an archaeal SKDH.
Publication types
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Alcohol Oxidoreductases / chemistry*
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Alcohol Oxidoreductases / genetics
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Alcohol Oxidoreductases / isolation & purification
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Alcohol Oxidoreductases / metabolism*
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Archaeoglobus fulgidus / enzymology*
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Cloning, Molecular
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Dimerization
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Enzyme Stability
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Half-Life
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Hydrogen-Ion Concentration
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Models, Molecular
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Molecular Weight
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NAD / metabolism
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NADP / metabolism
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Potassium Chloride
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Protein Conformation
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Recombinant Proteins / chemistry
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Shikimic Acid / analogs & derivatives*
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Shikimic Acid / metabolism
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Sodium Chloride
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Substrate Specificity
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Temperature
Substances
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Recombinant Proteins
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NAD
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3-dehydroshikimate
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Shikimic Acid
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Sodium Chloride
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NADP
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Potassium Chloride
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Alcohol Oxidoreductases
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Shikimate dehydrogenase