Identification, sequencing and mutagenesis of the gene for a D-carbamoylase from Agrobacterium radiobacter.
A clone positive for D-carbamoylase activity (2.7 kb HindIII-BamHI DNA fragment) was obtained by screening a genomic library of Agrobacterium radiobacter in Escherichia coli. This DNA fragment contains an open reading frame of 912 bp which is predicted to encode a peptide of 304 amino acids with a calculated molecular mass of 34247 Da. The D-carbamoylase gene, named cauA, was placed under the control of T7 RNA-dependent promoter and expressed in E. coli BL21(DE3). After induction with isopropyl-thio-beta-D-galactopyranoside, the synthesis of D-carbamoylase in E. coli reached about 40% of the total protein. The expressed protein was shown to possess a molecular mass, on SDS-PAGE, of 36 kDa and showed an enhanced stability with respect to that of the wild-type enzyme derived from A. radiobacter. Site-directed mutagenesis experiments allowed us to establish that a Pro14-->Leu14 exchange leads to an inactive enzyme species, while a Cys279-->Ser279 exchange did not impair the functional properties of the enzyme.
Buson
A
A
CRIBI Biotechnology Centre, University of Padua, Italy. albe@civ.bio.unipd.it
Negro
A
A
Grassato
L
L
Tagliaro
M
M
Basaglia
M
M
Grandi
C
C
Fontana
A
A
Nuti
M P
MP
D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D002265:Carboxylic Ester Hydrolases; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D012231:Rhizobium / Q000201:enzymology* / Q000235:genetics*; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D002265:Carboxylic Ester Hydrolases; D000581:Amidohydrolases; C080477:N-carbamoyl-D-amino acid amidohydrolase