Molecular characterisation of formamidase from Methylophilus methylotrophus.
A 3.2-kbp PstI fragment of DNA encoding formamidase from the methylotrophic bacterium Methylophilus methylotrophus which had previously been cloned (pNW3) [Wyborn, N.R., Scherr, D.J. & Jones, C.W. (1994) Microbiology 140, 191-195], was subcloned as a 2.3 kbp HindIII fragment (pNW323). Nucleotide sequencing showed that the subclone contained two genes which encoded formamidase (fmdA) and a possible regulatory protein (fmdB). Predicted molecular masses for FmdA and FmdB were 44438 Da (compared with approximately 44500 Da by electrospray mass spectrometry and 51000 Da by SDS/PAGE of the purified enzyme) and 12306 Da, respectively. The derived amino acid sequence of formamidase was supported by N-terminal amino acid sequencing of the enzyme and of proteolytic fragments prepared from it using V8 endoproteinase and was 57% similar to that of the acetamidase from Mycobacterium smegmatis. The structural similarities between these two enzymes, and their existence as a separate class of bacterial amidase, were confirmed by immunological investigations.
Wyborn
N R
NR
Department of Biochemistry, University of Leicester, England.
Mills
J
J
Williams
S G
SG
Jones
C W
CW
D000581:Amidohydrolases / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000641:Ammonia / Q000494:pharmacology; D015153:Blotting, Western; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames / Q000235:genetics; D015183:Restriction Mapping; D016415:Sequence Alignment; D017421:Sequence Analysis; D016335:Zinc Fingers
D003062:Codon; D000641:Ammonia; D000581:Amidohydrolases; C101846:formamidase