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Biochemical and Structural Characterization of a Thermophilc L-Arabinose Isomerase from Geobacillus kaustophilus
New Biotechnology · Volume 31S · July 2014
BIOCATALYSIS
PA-18
PA-19
Biochemical and Structural Characterization of a Thermophilc L-Arabinose Isomerase from Geobacillus kaustophilus
Functional Characterization of Putative UDP-Glucose 4-Epimerase (TM0509) from the Hyperthermophilic Eubacterium Thermotoga maritima
Dong-Woo Lee 1,∗ , Yong-Jik Lee 1 , Jin Myung Choi 2 , Sun-Mi Shin 1 , Sang-Jae Lee 3 , Han-Seung Lee 3 , Sang Jun Lee 4 , Sung Haeng Lee 2
Sun-Mi Shin 1,∗ , Jin Myung Choi 2 , Yong-Jik Lee 1 , Sang-Jae Lee 3 , Sang Jun Lee 4 , Sung Haeng Lee 2 , Dong-Woo Lee 5
1
1
2
2
Kyungpook National University Chosun University 3 Silla University 4 KRIBB
Thermophilic L-Arabinose isomerase (AI), that catalyzes the interconversion of L-arabinose to L-ribulose, can also isomerize D-galactose to D-tagatose as a natural sugar substitute, which is of commercial interest in the food and healthcare industries.Recently, biochemical and mutational studies revealed that unlike mesophilic AIs, thermophilic AIs showed the distinct metal dependence for their catalytic activity and thermostability at elevated temperatures. However, it still remains unclear how mesophilic and thermophilic AIs showed different substrate preferences and metal requirements at molecular levels. Herein we characterized a thermophilic AI from Geobacillus kaustophilus (GKAI) and presented the first crystal structures of the apo and holo ˚ respecforms of GKAI by X-ray crystallography to 2.40 and 2.30 A, tively. We also determined the crystal structure of holo enzyme ˚ resolution.In bound to L-arabitol as a substrate analog at 2.25 A combination with biochemical and site-directed mutagenesis studies, the structures identified the structural elements of metal binding and substrate recognition.In comparison with the crystal structures of Escherichia coli AI (ECAI) as a mesophilic counterpart, the GKAI structures revealed quite conserved structural features for substrate and metal binding, except forsubtle interactions of a few polar residues with water molecules near the substrate binding region.Our comparative analysis proposes a metal-mediated substrate binding model for the isomerization reaction at elevated temperatures, providing a versatile strategy to engineer the promiscuity of substrate specificity for sugar isomerases as well as thermostability for mechanistic studies and industrial applications.
Kyungpook National University Chosun University 3 Silla University 4 Korea Research Institute of Bioscience and Biotechnology 5 Kyungpook National Univ
UDP-glucose 4-epimerase (GalE; EC 5.1.3.2) catalyzes the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal), which is a pivotal step in the Leloir pathway for galactose metabolism. Although GalEs are widely distributed in Bacteria and Eukaryotes, there is little information on hyperthermophilic GalE. Herein we cloned and overexpressed the TM0509 gene encoding a putative GalE from Thermotoga maritima (TMGalE) as a fusion protein containing an N-terminal hexa-histidine sequence in Escherichia coli. This gene encodes a 309-amino acid protein with a calculated molecular weight of 34899 and a theoretical pI of 5.72.The recombinant protein was purified to homogeneity by heat precipitation, Ni2+ affinity chromatography followed by size-exclusion chromatography. The native enzyme was estimated to be a homodimer with a molecular mass of 70 kDa. The recombinant TMGalE could reversibly catalyze the epimerization of UDP-Gal and UDP-Glc in the presence of NAD+ at elevated temperatures. The apparent optimal temperature and pH for epimerization activity were 85 ◦ C and pH 7.0, respectively. In order to further characterize TMGalE at molecular levels, we determined ˚ resolution, but not only the crystal structure of TMGalE at 1.9 A also the co-crystal structure of TMGalE bound to UDP-glucose at ˚ resolution. These biochemical and structural data showed 2.0 A that TM0509 is an UDP-galactose 4-epimerase involved in galactose metabolism, which is the first detailed characterization of a thermostable GalE from hyperthermophilic bacterium. http://dx.doi.org/10.1016/j.nbt.2014.05.1805
http://dx.doi.org/10.1016/j.nbt.2014.05.1804 PA-20 Microscale tools for evaluating the biological and process options of alkane biooxidations Johannes Kolmar 1,∗ , Frank Baganz 1 , Philip Engel 2 1
2
University College London Evonik Industries AG
The direct -oxyfunctionalisation of aliphatic alkanes in a regio- and chemoselective manner remains difficult to perform by industrial organic chemistry. Monooxygenases such as the AlkB enzyme complex from Pseudomonas putida efficiently catalyse these readily available substrates to primary fatty alcohols and acids under mild conditions. These are of considerable interest as potential intermediates in the chemical, pharmaceutical and cosmetics industry. S84
www.elsevier.com/locate/nbt
BIOCATALYSIS
PA-18
PA-19
Biochemical and Structural Characterization of a Thermophilc L-Arabinose Isomerase from Geobacillus kaustophilus
Functional Characterization of Putative UDP-Glucose 4-Epimerase (TM0509) from the Hyperthermophilic Eubacterium Thermotoga maritima
Dong-Woo Lee 1,∗ , Yong-Jik Lee 1 , Jin Myung Choi 2 , Sun-Mi Shin 1 , Sang-Jae Lee 3 , Han-Seung Lee 3 , Sang Jun Lee 4 , Sung Haeng Lee 2
Sun-Mi Shin 1,∗ , Jin Myung Choi 2 , Yong-Jik Lee 1 , Sang-Jae Lee 3 , Sang Jun Lee 4 , Sung Haeng Lee 2 , Dong-Woo Lee 5
1
1
2
2
Kyungpook National University Chosun University 3 Silla University 4 KRIBB
Thermophilic L-Arabinose isomerase (AI), that catalyzes the interconversion of L-arabinose to L-ribulose, can also isomerize D-galactose to D-tagatose as a natural sugar substitute, which is of commercial interest in the food and healthcare industries.Recently, biochemical and mutational studies revealed that unlike mesophilic AIs, thermophilic AIs showed the distinct metal dependence for their catalytic activity and thermostability at elevated temperatures. However, it still remains unclear how mesophilic and thermophilic AIs showed different substrate preferences and metal requirements at molecular levels. Herein we characterized a thermophilic AI from Geobacillus kaustophilus (GKAI) and presented the first crystal structures of the apo and holo ˚ respecforms of GKAI by X-ray crystallography to 2.40 and 2.30 A, tively. We also determined the crystal structure of holo enzyme ˚ resolution.In bound to L-arabitol as a substrate analog at 2.25 A combination with biochemical and site-directed mutagenesis studies, the structures identified the structural elements of metal binding and substrate recognition.In comparison with the crystal structures of Escherichia coli AI (ECAI) as a mesophilic counterpart, the GKAI structures revealed quite conserved structural features for substrate and metal binding, except forsubtle interactions of a few polar residues with water molecules near the substrate binding region.Our comparative analysis proposes a metal-mediated substrate binding model for the isomerization reaction at elevated temperatures, providing a versatile strategy to engineer the promiscuity of substrate specificity for sugar isomerases as well as thermostability for mechanistic studies and industrial applications.
Kyungpook National University Chosun University 3 Silla University 4 Korea Research Institute of Bioscience and Biotechnology 5 Kyungpook National Univ
UDP-glucose 4-epimerase (GalE; EC 5.1.3.2) catalyzes the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal), which is a pivotal step in the Leloir pathway for galactose metabolism. Although GalEs are widely distributed in Bacteria and Eukaryotes, there is little information on hyperthermophilic GalE. Herein we cloned and overexpressed the TM0509 gene encoding a putative GalE from Thermotoga maritima (TMGalE) as a fusion protein containing an N-terminal hexa-histidine sequence in Escherichia coli. This gene encodes a 309-amino acid protein with a calculated molecular weight of 34899 and a theoretical pI of 5.72.The recombinant protein was purified to homogeneity by heat precipitation, Ni2+ affinity chromatography followed by size-exclusion chromatography. The native enzyme was estimated to be a homodimer with a molecular mass of 70 kDa. The recombinant TMGalE could reversibly catalyze the epimerization of UDP-Gal and UDP-Glc in the presence of NAD+ at elevated temperatures. The apparent optimal temperature and pH for epimerization activity were 85 ◦ C and pH 7.0, respectively. In order to further characterize TMGalE at molecular levels, we determined ˚ resolution, but not only the crystal structure of TMGalE at 1.9 A also the co-crystal structure of TMGalE bound to UDP-glucose at ˚ resolution. These biochemical and structural data showed 2.0 A that TM0509 is an UDP-galactose 4-epimerase involved in galactose metabolism, which is the first detailed characterization of a thermostable GalE from hyperthermophilic bacterium. http://dx.doi.org/10.1016/j.nbt.2014.05.1805
http://dx.doi.org/10.1016/j.nbt.2014.05.1804 PA-20 Microscale tools for evaluating the biological and process options of alkane biooxidations Johannes Kolmar 1,∗ , Frank Baganz 1 , Philip Engel 2 1
2
University College London Evonik Industries AG
The direct -oxyfunctionalisation of aliphatic alkanes in a regio- and chemoselective manner remains difficult to perform by industrial organic chemistry. Monooxygenases such as the AlkB enzyme complex from Pseudomonas putida efficiently catalyse these readily available substrates to primary fatty alcohols and acids under mild conditions. These are of considerable interest as potential intermediates in the chemical, pharmaceutical and cosmetics industry. S84
www.elsevier.com/locate/nbt