Phylogenetic analysis and biochemical characterization of a thermostable dihydropyrimidinase from alkaliphilic Bacillus sp. TS-23.
Two degenerate primers established from the alignment of highly conserved amino acid sequences of bacterial dihydropyrimidinases (DHPs) were used to amplify a 330-bp gene fragment from the genomic DNA of Bacillus sp. TS-23 and the amplified DNA was successfully used as a probe to clone a dhp gene from the strain. The open reading frame of the gene consisted of 1422 bp and was deduced to contain 472 amino acids with a molecular mass of 52 kDa. The deduced amino acid sequence exhibited greater than 45% identity with that of prokaryotic D-hydantoinases and eukaryotic DHPs. Phylogenetic analysis showed that Bacillus sp. TS-23 DHP is grouped together with Bacillus stearothermophilus D-hydantoinase and related to dihydroorotases and allantoinases from various organisms. His6-tagged DHP was over-expressed in Escherichia coli and purified by immobilized metal affinity chromatography to a specific activity of 3.46 U mg(-1) protein. The optimal pH and temperature for the purified enzyme were 8.0 and 60 degrees C, respectively. The half-life of His6-tagged DHP was 25 days at 50 degrees C. The enzyme activity was stimulated by Co2+ and Mn2+ ions. His6-tagged DHP was most active toward dihydrouracil followed by hydantoin derivatives. The catalytic efficiencies (kcat/Km) of the enzyme for dihydrouracil and hydantoin were 2.58 and 0.61 s(-1) mM(-1), respectively.
Lin
Long-Liu
LL
Department of Applied Chemistry, National Chiayi University, Chiayi 60083, Taiwan.
Hsu
Wen-Hwei
WH
Hsu
Wei-Yi
WY
Kan
Shu-Chen
SC
Hu
Hui-Yu
HY
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003067:Coenzymes / Q000494:pharmacology; D004269:DNA, Bacterial / Q000235:genetics; D004080:Dihydroorotase / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006827:Hydantoins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D014498:Uracil / Q000031:analogs & derivatives / Q000378:metabolism
D003067:Coenzymes; D004269:DNA, Bacterial; D006827:Hydantoins; D008670:Metals; D011994:Recombinant Proteins; C007419:dihydrouracil; D014498:Uracil; D000581:Amidohydrolases; C020047:dihydropyrimidinase; D004080:Dihydroorotase; C019659:allantoinase