A MANGANESE-DEPENDENT DIOXYGENASE FROM ARTHROBACTER-GLOBIFORMIS CM-2 BELONGS TO THE MAJOR EXTRADIOL DIOXYGENASE FAMILY

Almost all bacterial ring cleavage dioxygenases contain iron as the ca talytic metal center. We report here the first available sequence for a manganese-dependent 3,4-dihydroxyphenylacetate (3,4-DHPA) 2,3-dioxyg enase and its further characterization. This manganese-dependent extra diol dioxygenase from Arthrobacter globiformis CM-2, unlike iron-depen dent extradiol dioxygenases, is not inactivated by hydrogen peroxide. Also, ferrous ions,,which activate iron extradiol dioxygenases, inhibi t 3,4-DHPA 2,3-dioxygenase. The gene encoding 3,4-DHPA 2,3-dioxygenase , mndD, was identified from an A. globiformis CM-2 cosmid library. mnd D aas subcloned as a 2.0-kb SmaI fragment in pUC18, from which mangane se-dependent extradiol dioxygenase activity was expressed at high leve ls in Escherichia coli. The mndD open reading frame was identified by comparison with the known N-terminal amino acid sequence of purified m anganese-dependent 3,4-DHPA 2,3-dioxygenase. Fourteen of 18 amino acid s conserved in members of the iron-dependent extradiol dioxygenase fam ily are also conserved in the manganese-dependent 3,4-DHPA 2,3-dioxyge nase (MndD). Thus, MndD belongs to the extradiol family of dioxygenase s and may share a common ancestry with the iron-dependent extradiol di oxygenases; We propose the revised consensus primary sequence ,N,R)X(H ,A)XXXXXXX(L,I,V,M,F)YXX(D,E,T,N,A)PX(G,P) X{2,3}E for this family. (N umbers in brackets indicate a gap of two or three residues at this poi nt in the sequence.) The suggested common ancestry is also supported b y sequence obtained froth genes flanking mndD, which share significant sequence identity with xylJ and xylG from Pseudomonas putida.