Protocatechuate 4,5-dioxygenase fromComamonas testosteroniT-2: biochemical and molecular properties of a new subgroup within class III of extradiol dioxygenases.

Comamonas testosteroniT-2 degraded at least eight aromatic compounds via protocatechuate (PCA), whose extradiol ring cleavage to 2-hydroxy-4-carboxymuconate semialdehyde (HCMS) was catalysed by PCA 4,5-dioxygenase (PmdAB). This inducible, heteromultimeric enzyme was purified. It contained two subunits, a (PmdA) and ß (PmdB), and the molecular masses of the denatured proteins were 18 kDa and 31 kDa, respectively. PCA was converted stoichiometrically to HCMS with an apparentKm of 55 µM and at a maximum velocity of 1.5 µkat. Structure-activity-relationship analysis by testing 16 related compounds as substrate for purified PmdAB revealed an absolute requirement for the vicinal diol and for the carboxylate group of PCA. Besides PCA, only 5'-hydroxy-PCA (gallate) induced oxygen uptake. The N-terminal amino acid sequence of each subunit was identical to the corresponding sequences inC. testosteroniBR6020, which facilitated sequencing of thepmdABgenes in strain T-2. Small differences in the amino acid sequence had significant effects on enzyme stability. Several homologues ofpmdABwere found in sequence databases. Residues involved in substrate binding are highly conserved among the homologues. Their sequences grouped within the class III extradiol dioxygenases. Based on our biochemical and genetic analyses, we propose a new branch of the heteromultimeric enzymes within that class. [ABSTRACT FROM AUTHOR]