ESI- and MALDI-MS analysis of cyclohexanone monooxygenase from Acinetobacter NCIB 9871

Recombinant and native forms of cyclohexanone monooxygenase (CMO) from Acin etobacter NCIB 9871 were analyzed by mass spectrometry to probe ambiguities arising from the presence of multiple DNA sequences for the enzyme in GenB ank. A CMO gene corresponding exactly to the nucleotide sequence described by Iwaki ct al. (10) was amplified from genomic DNA, cloned into pET15b, an d the recombinant protein purified from a bacterial expression system. Elec trospray mass spectrometry of both the recombinant material and the native form of CMO isolated from Acinetobacter yielded molecular weights within 0. 01% of those predicted from the translated gene sequence of Iwaki ct al. (1 0), Trypsin and chymotrypsin digests of native CMO, analyzed by electrospra y and MALDI mass spectrometry, provided greater than 97% coverage of the pr otein and confirmed the presence of specific peptide sequences predicted by the Iwaki sequence alone. Therefore, the primary sequence of native Acinet obacter CMO is identical to the gene sequence for chnB deposited under acce ssion number AB006902. (C) 2001 Academic Press.