Characterization and phylogenetic analysis of a thermostable N-carbamoyl- l-amino acid amidohydrolase from Bacillus kaustophilus CCRC11223.

Abstract

A thermostable N-carbamoyl- l-amino acid amidohydrolase ( l-N-carbamoylase) gene composed of an 1,230-bp ORF encoding a 44.3-kDa protein was cloned from the thermophile Bacillus kaustophilus CCRC11223. This l-N-carbamoylase contained six cysteine residues that form three disulfide bridges. The purified l-N-carbamoylase was stringently l-specific and exhibited high activity in the hydrolysis of N-carbamoyl- l-homophenylalanine. N-carbamoyl derivatives of beta-alanine, beta-aminoisobutyric acids, l-tryptophan, and d-specific amino acids were not recognized as substrates. The l-N-carbamoylase required the divalent metal ions Mn(2+), Co(2+), and Ni(2+) for increasing activity. The pH and temperature optima of the enzyme were pH 7.4 and 70 degrees C, respectively. This enzyme was completely thermostable at 50 degrees C for 36 days in the presence of d- and/or l-specific substrates. Phylogenetic analysis of the available amino acid sequences of N-carbamoyl and N-acyl amino acid amidohydrolases from the three main kingdoms of life showed that they can be divided into four distinct families. The B. kaustophilus enzyme could be classified into the family of l-N-carbamoylases and some beta-ureidopropionases, but did not hydrolyze beta-ureidopropionates.

• Authors: Hui-Yu Hu, Wen-Hwei Hsu, Hungchien Roger Chien
• Journal: Archives of microbiology (Arch Microbiol) Vol. 179 Issue 4 Pg. 250-7 (Apr 2003) ISSN: 0302-8933 [Print] Germany
• PMID: 12605292 (Publication Type: Comparative Study, Journal Article)

Chemical References

• Metals
• Recombinant Proteins
• Amidohydrolases
• N-carbamoylamino acid amidohydrolase
• Alanine

Topics

• Alanine (chemistry)
• Amidohydrolases (chemistry, genetics, metabolism)
• Amino Acid Sequence
• Bacillus (enzymology, genetics)
• Cloning, Molecular
• Escherichia coli (metabolism)
• Genes, Bacterial
• Hydrogen-Ion Concentration
• Metals
• Molecular Sequence Data
• Molecular Weight
• Recombinant Proteins (metabolism)
• Sequence Alignment
• Substrate Specificity
• Temperature