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PMID: 12374829 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

The first archaeal ATP-dependent glucokinase, from the hyperthermophilic crenarchaeon Aeropyrum pernix, represents a monomeric, extremely thermophilic ROK glucokinase with broad hexose specificity.

Journal of bacteriology ·Vol. 184 ·No. 21 ·2002-11-00 ·Pages 5955-65

Hansen T, Reichstein B, Schmid R, Schönheit P

Abstract

An ATP-dependent glucokinase of the hyperthermophilic aerobic crenarchaeon Aeropyrum pernix was purified 230-fold to homogeneity. The enzyme is a monomeric protein with an apparent molecular mass of about 36 kDa. The apparent K(m) values for ATP and glucose (at 90 degrees C and pH 6.2) were 0.42 and 0.044 mM, respectively; the apparent V(max) was about 35 U/mg. The enzyme was specific for ATP as a phosphoryl donor, but showed a broad spectrum for phosphoryl acceptors: in addition to glucose, which showed the highest catalytic efficiency (k(cat)/K(m)), the enzyme also phosphorylates glucosamin, fructose, mannose, and 2-deoxyglucose. Divalent cations were required for maximal activity: Mg(2+), which was most effective, could partially be replaced with Co(2+), Mn(2+), and Ni(2+). The enzyme had a temperature optimum of at least 100 degrees C and showed significant thermostability up to 100 degrees C. The coding function of open reading frame (ORF) APE2091 (Y. Kawarabayasi, Y. Hino, H. Horikawa, S. Yamazaki, Y. Haikawa, K. Jin-no, M. Takahashi, M. Sekine, S. Baba, A. Ankai, H. Kosugi, A. Hosoyama, S. Fukui, Y. Nagai, K. Nishijima, H. Nakazawa, M. Takamiya, S. Masuda, T. Funahashi, T. Tanaka, Y. Kudoh, J. Yamazaki, N. Kushida, A. Oguchi, and H. Kikuchi, DNA Res. 6:83-101, 145-152, 1999), previously annotated as gene glk, coding for ATP-glucokinase of A. pernix, was proved by functional expression in Escherichia coli. The purified recombinant ATP-dependent glucokinase showed a 5-kDa higher molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but almost identical kinetic and thermostability properties in comparison to the native enzyme purified from A. pernix. N-terminal amino acid sequence of the native enzyme revealed that the translation start codon is a GTG 171 bp downstream of the annotated start codon of ORF APE2091. The amino acid sequence deduced from the truncated ORF APE2091 revealed sequence similarity to members of the ROK family, which comprise bacterial sugar kinases and transcriptional repressors. This is the first report of the characterization of an ATP-dependent glucokinase from the domain of Archaea, which differs from its bacterial counterparts by its monomeric structure and its broad specificity for hexoses.

MeSH Terms
Adenosine Triphosphate/metabolism Amino Acid Sequence Catalysis Cloning, Molecular Desulfurococcaceae/enzymology Enzyme Stability Escherichia coli Gene Expression Glucokinase/genetics,isolation & purification,metabolism Molecular Sequence Data Molecular Weight Open Reading Frames Recombinant Fusion Proteins/genetics,isolation & purification,metabolism Sequence Homology, Amino Acid Substrate Specificity Temperature
Chemicals
Recombinant Fusion Proteins Adenosine Triphosphate Glucokinase
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Hansen Thomas
Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, D-24118 Kiel, Am Botanischen Garten 1-9, Germany.
Reichstein Bianca
Schmid Roland
Schönheit Peter
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/JB.184.21.5955-5965.2002
Published
2002-11-00
Pages
5955-65
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC135380
Subset
IM
External Links
PubMed source DOI: 10.1128/JB.184.21.5955-5965.2002 PMC Full Text View journal details
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