Substrate and binding specificities of bacterial polyhydroxybutyrate depolymerases

The substrate specificities of three extracellular polyhydroxybutyrate (PHB ) depolymerases from Alcaligenes faecalis (PhaZ(Afa)), Pseudomonas stutzeri (PhaZ(Pst)), and Comamonas acidovorans (PhaZ(Cac)), which are grouped into types A and B based on the position of a lipase box sequence in the cataly tic domain, were examined for films of 12 different aliphatic polyesters. E ach of these PHB depolymerases used was capable of hydrolyzing poly(3-hydro xybutyrate) (P(3HB)), poly(3-hydroxypropionate) (P(3HP)), poly(4-hydroxybut yrate) (P(4HB)), poly(ethylene succinate) (PESU), and poly(ethylene adipate ) (PEA) but could not hydrolyze another seven polyesters. In addition, the binding characteristics of substrate binding domains from PhaZ(Afa), PhaZ(C ac), and PHB depolymerase from Comamonas testosteroni (PhaZ(Cte)) were stud ied by using fusions with glutathione S-transferase (GST). All of fusion pr oteins adsorbed strongly on the surfaces of polyester granules of P(3HB), P (3HP), and poly(2-hydroxypropionate) (P(2HP)) which was not hydrolyzed by t he PHB depolymerases used in this study, while they did not bind on Avicel and chitin granules. The adsorption kinetics of the fusion proteins to the surface of P(3HB) and P(2HP) granules were found to obey the Langmuir isoth erm. The cross-area per molecule of fusion protein bound to P(3HB) granules was estimated to be 12 +/- 4 nm(2)/molecule. It has been suggested that th e active sites in catalytic domains of PHB depolymerases have a similar con formational structure, and that several amino acids in substrate-binding do mains of PHB depolymerases interact specifically with the surface of polyes ters. (C) 1999 Elsevier Science B.V. All rights reserved.