MOLECULAR CHARACTERIZATION OF FORMAMIDASE FROM METHYLOPHILUS-METHYLOTROPHUS

A 3.2-kbp PstI fragment of DNA encoding formamidase from the methylotr ophic bacterium Methylophilus methylotrophus which had previously been cloned (pNW3) [Wyborn, N. R., Scherr, D. J. & Jones, C. W. (1994) Mic robiology 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 prote in (fmdB). Predicted molecular masses for FmdA and FmdB were 44438 Da (compared with approximately 44500 Da by electrospray mass spectrometr y and 51000 Da by SDS/PAGE of the purified enzyme) and 12306 Da, respe ctively. The derived amino acid sequence of formamidase was supported by N-terminal amino acid sequencing of the enzyme and of proteolytic f ragments prepared from it using V8 endoproteinase and was 57% similar to that of the acetamidase from Mycobacterium smegmatis, The structura l similarities between these two enzymes, and their existence as a sep arate class of bacterial amidase, were confirmed by immunological inve stigations.