title: Creation of metal-independent hyperthermophilic L-arabinose isomerase by homologous recombination.
abstract: Hyperthermophilic L-arabinose isomerases (AIs) are useful in the commercial production of D-tagatose as a low-calorie bulk sweetener. Their catalysis and thermostability are highly dependent on metals, which is a major drawback in food applications. To study the role of metal ions in the thermostability and catalysis of hyperthermophilic AI, four enzyme chimeras were generated by PCR-based hybridization to replace the variable N- and C-terminal regions of hyperthermophilic Thermotoga maritima AI (TMAI) and thermophilic Geobacillus stearothermophilus AI (GSAI) with those of the homologous mesophilic Bacillus halodurans AI (BHAI). Unlike Mn(2+)-dependent TMAI, the GSAI- and TMAI-based hybrids with the 72 C-terminal residues of BHAI were not metal-dependent for catalytic activity. By contrast, the catalytic activities of the TMAI- and GSAI-based hybrids containing the N-terminus (residues 1-89) of BHAI were significantly enhanced by metals, but their thermostabilities were poor even in the presence of Mn(2+), indicating that the effects of metals on catalysis and thermostability involve different structural regions. Moreover, in contrast to the C-terminal truncate (Δ20 residues) of GSAI, the N-terminal truncate (Δ7 residues) exhibited no activity due to loss of its native structure. The data thus strongly suggest that the metal dependence of the catalysis and thermostability of hyperthermophilic AIs evolved separately to optimize their activity and thermostability at elevated temperatures. This may provide effective target regions for engineering, thereby meeting industrial demands for the production of d-tagatose.
authors: CJ Foods R & D, CJ Cheiljedang Corporation, Seoul 100-749, Korea.
authors: Young-Ho
authors: YH
authors: Hong
authors: Dong-Woo
authors: DW
authors: Lee
authors: Yu-Ryang
authors: YR
authors: Pyun
authors: Sung Haeng
authors: SH
authors: Lee
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006601:Hexoses / Q000096:biosynthesis; D059765:Homologous Recombination; D006358:Hot Temperature; D008345:Manganese / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013549:Sweetening Agents
chemical_list: D006601:Hexoses; D008670:Metals; D011994:Recombinant Proteins; D013549:Sweetening Agents; D008345:Manganese; D019747:Aldose-Ketose Isomerases; C019715:L-arabinose isomerase; C030192:tagatose