Purification and some properties of lignostilbene-alpha, beta-dioxygenase isozyme IV from Pseudomonas paucimobilis TMY1009.

Lignostilbene-alpha, beta-dioxygenase isozyme IV was purified from ultrasonic extracts of Pseudomonas paucimobilis TMY1009 through four steps of column chromatography. The fraction obtained gave a single band on SDS-PAGE and a single peak on reversed-phase HPLC and DEAE-HPLC. The specific activity and Km of purified isozyme IV were 110 mu kat/g and 4.2 microM for 4.4'-dihydroxy-3,3'-dimethoxystilbene, 150 mu kat/g and 3.3 microM for 4,2'-dihydroxy-3,3'-dimethoxy-5'-(2"-carboxyvinyl)stilbene. The molecular mass of intact isozyme IV was estimated to be 94 kDa by gel permeation chromatography, and that of its subunits was 52 kDa by SDS-PAGE under denaturing conditions. The N-terminal amino acid sequence of isozyme IV differed slightly from that of other isozymes. Isozyme IV seemed to be composed of two identical subunits, gamma gamma.