New gene cluster from the thermophile Bacillus fordii MH602 in the conversion of DL-5-substituted hydantoins to L-amino acids.
The thermophile Bacillus fordii MH602 was screened for stereospecifically hydrolyzing DL-5-substituted hydantoins to L-alpha-amino acids. Since the reaction at higher temperature, the advantageous for enhancement of substrate solubility and for racemization of DL-5-substituted hydantoins during the conversion were achieved. The hydantoin metabolism gene cluster from thermophile was firstly reported in this paper. The genes involved in hydantoin utilization (hyu) were isolated on an 8.2 kb DNA fragment by Restriction Site-dependent PCR, and six ORFs were identified by DNA sequence analysis. The hyu gene cluster contained four genes with novel cluster organization characteristics: the hydantoinase gene hyuH, putative transport protein hyuP, hyperprotein hyuHP, and L-carbamoylase gene hyuC. The hyuH and hyuC genes were heterogeneously expressed in E. coli. The results indicated that hyuH and hyuC are involved in the conversion of DL-5-substituted hydantoins to an N-carbamyl intermediate that is subsequently converted to L-alpha-amino acids. Hydantoinase and carbamoylase from B. fordii MH602 comparing respectively with reported hydantoinase and carbamoylase showed the highest identities of 71% and 39%. The novel cluster organization characteristics and the difference of the key enzymes between thermopile B. fordii MH602 and other mesophiles were presumed to be related to the evolutionary origins of concerned metabolism.
Mei
Yan-Zhen
YZ
College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing, 210009, Republic of China.
Wan
Yong-Min
YM
He
Bing-Fang
BF
Ying
Han-Jie
HJ
Ouyang
Ping-Kai
PK
D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism; D000596:Amino Acids / Q000096:biosynthesis* / Q000737:chemistry; D001407:Bacillus / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D005798:Genes, Bacterial; D006358:Hot Temperature; D006827:Hydantoins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D000596:Amino Acids; D002352:Carrier Proteins; D004269:DNA, Bacterial; D006827:Hydantoins; D011994:Recombinant Proteins; D000581:Amidohydrolases; C104561:N-carbamoyl-L-amino-acid hydrolase; C020047:dihydropyrimidinase