2,4 '-Dihydroxyacetophenone dioxygenase (EC 1.13.11.41) from Alcaligenes sp 4HAP: a novel enzyme with an atypical dioxygenase sequence

2,4'-Dihydroxyacetophenone dioxygenase (EC 1.13.11.41) was purified to homo geneity from Alcaligenes sp. 4HAP grown on 4-hydroxyacetophenone. Measureme nts of the M-r of the native enzyme ranged from 81600 to 87000, whereas val ues of 21000 and 20379 were given by SDS/PAGE and electrospray MS respectiv ely. The enzyme is a homotetramer and contains one atom of iron per molecul e of enzyme. From C- and N-terminal analyses, primers for PCR were designed and the dad gene cloned and sequenced. The predicted amino acid sequence o f dad, deduced from the nucleotide sequence, confirms the N-terminal amino acid sequencing data and contains the sequence of an internal tryptic pepti de. It gave a calculated M-r of 20364. The gene was expressed in Escherichi a coli and yielded active enzyme. The derived amino acid sequence does not show significant similarity to other dioxygenases or any strong similarity to protein sequences presently available in the databases.