title: Thermotoga neapolitana adenylate kinase is highly active at 30 degrees C.
abstract: The adenylate kinase (AK) gene from Thermotoga neapolitana, a hyperthermophilic bacterium, was cloned and overexpressed in Escherichia coli, and the recombinant enzyme was biochemically characterized. The T. neapolitana AK (TNAK) sequence indicates that this enzyme belongs to the long bacterial AKs. TNAK contains the four cysteine residues that bind Zn(2+) in all Gram-positive AKs and in a few other Zn(2+)-containing bacterial AKs. Atomic emission spectroscopy and titration data indicate a content of 1 mol of Zn(2+)/mol of recombinant TNAK. The EDTA-treated enzyme has a melting temperature (T (m)=93.5 degrees C) 6.2 degrees C below that of the holoenzyme (99.7 degrees C), identifying Zn(2+) as a stabilizing feature in TNAK. TNAK is a monomeric enzyme with a molecular mass of approx. 25 kDa. TNAK displays V (max) and K (m) values at 30 degrees C identical with those of the E. coli AK at 30 degrees C, and displays very high activity at 80 degrees C, with a specific activity above 8000 units/mg. The unusually high activity of TNAK at 30 degrees C makes it an interesting model to test the role of enzyme flexibility in activity.
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing 48824, USA. vieille@msu.edu
authors.forename: Claire
authors.initials: C
authors.lastname: Vieille
authors.forename: Harini
authors.initials: H
authors.lastname: Krishnamurthy
authors.forename: Hyung-Hwan
authors.initials: HH
authors.lastname: Hyun
authors.forename: Alexei
authors.initials: A
authors.lastname: Savchenko
authors.forename: Honggao
authors.initials: H
authors.lastname: Yan
authors.forename: J Gregory
authors.initials: JG
authors.lastname: Zeikus
mesh_terms: D000263:Adenylate Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001051:Apoenzymes; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016965:Gram-Negative Anaerobic Straight, Curved, and Helical Rods / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
chemical_list: D001051:Apoenzymes; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D000263:Adenylate Kinase; D015032:Zinc