A thermostable shikimate 5-dehydrogenase from the archaeon Archaeoglobus fulgidus.
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.
Lim
Sierin
S
Biomedical Engineering Interdepartmental Program, 7523 Boelter Hall, University of California, 1602 Molecular Sciences Bldg., Los Angeles, CA 90095-1489, USA.
Schröder
Imke
I
Monbouquette
Harold G
HG
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019611:Archaeoglobus fulgidus / Q000201:enzymology*; D003001:Cloning, Molecular; D019281:Dimerization; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D011189:Potassium Chloride; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012765:Shikimic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D012965:Sodium Chloride; D013379:Substrate Specificity; D013696:Temperature
D011994:Recombinant Proteins; D009243:NAD; C096387:3-dehydroshikimate; D012765:Shikimic Acid; D012965:Sodium Chloride; D009249:NADP; D011189:Potassium Chloride; D000429:Alcohol Oxidoreductases; C022641:Shikimate dehydrogenase