title: Distinct metal dependence for catalytic and structural functions in the L-arabinose isomerases from the mesophilic Bacillus halodurans and the thermophilic Geobacillus stearothermophilus.
abstract: L-Arabinose isomerase (AI) catalyzes the isomerization of L-arabinose to L-ribulose. It can also convert d-galactose to d-tagatose at elevated temperatures in the presence of divalent metal ions. The araA genes, encoding AI, from the mesophilic bacterium Bacillus halodurans and the thermophilic Geobacillus stearothermophilus were cloned and overexpressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The purified enzymes are homotetramers with a molecular mass of 232 kDa and close amino acid sequence identity (67%). However, they exhibit quite different temperature dependence and metal requirements. B. halodurans AI has maximal activity at 50 degrees C under the assay conditions used and is not dependent on divalent metal ions. Its apparent K(m) values are 36 mM for L-arabinose and 167 mM for d-galactose, and the catalytic efficiencies (k(cat)/K(m)) of the enzyme were 51.4 mM(-1)min(-1) (L-arabinose) and 0.4 mM(-1)min(-1) (d-galactose). Unlike B. halodurans AI, G. stearothermophilus AI has maximal activity at 65-70 degrees C, and is strongly activated by Mn(2+). It also has a much higher catalytic efficiency of 4.3 mM(-1)min(-1) for d-galactose and 32.5 mM(-1)min(-1)for L-arabinose, with apparent K(m) values of 117 and 63 mM, respectively. Irreversible thermal denaturation experiments using circular dichroism (CD) spectroscopy showed that the apparent melting temperature of B. halodurans AI (T(m)=65-67 degrees C) was unaffected by the presence of metal ions, whereas EDTA-treated G. stearothermophilus AI had a lower T(m) (72 degrees C) than the holoenzyme (78 degrees C). CD studies of both enzymes demonstrated that metal-mediated significant conformational changes were found in holo G. stearothermophilus AI, and there is an active tertiary structure for G. stearothermophilus AI at elevated temperatures for its catalytic activity. This is in marked contrast to the mesophilic B. halodurans AI where cofactor coordination is not necessary for proper protein folding. The metal dependence of G. stearothermophilus AI seems to be correlated with their catalytic and structural functions. We therefore propose that the metal ion requirement of the thermophilic G. stearothermophilus AI reflects the need to adopt the correct substrate-binding conformation and the structural stability at elevated temperatures.
authors.affiliation: Department of Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea.
authors.forename: Dong-Woo
authors.initials: DW
authors.lastname: Lee
authors.forename: Eun-Ah
authors.initials: EA
authors.lastname: Choe
authors.forename: Seong-Bo
authors.initials: SB
authors.lastname: Kim
authors.forename: Soo-Hyun
authors.initials: SH
authors.lastname: Eom
authors.forename: Young-Ho
authors.initials: YH
authors.lastname: Hong
authors.forename: Sang-Jae
authors.initials: SJ
authors.lastname: Lee
authors.forename: Han-Seung
authors.initials: HS
authors.lastname: Lee
authors.forename: Dong-Yun
authors.initials: DY
authors.lastname: Lee
authors.forename: Yu-Ryang
authors.initials: YR
authors.lastname: Pyun
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000737:chemistry*; D001407:Bacillus / Q000201:enzymology / Q000378:metabolism*; D015153:Blotting, Western; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D007477:Ions; D007525:Isoelectric Focusing; D007700:Kinetics; D008345:Manganese / Q000737:chemistry; D008670:Metals; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature; D013997:Time Factors
chemical_list: D007477:Ions; D008670:Metals; D011994:Recombinant Proteins; D008345:Manganese; D019747:Aldose-Ketose Isomerases; C019715:L-arabinose isomerase