Investigation of the Enzymatic Cleavage of Diastereomeric Oligo(3-hydroxybutanoates) Containing Two to Eight HB Units. A Model for the Stereoselectivity of PHB Depolymerase from <i>Alcaligenes faecalis</i> T₁

Abstract

So far, it is known that various poly(3-hydroxybutanoate), PHB, depolymerases are able to degrade all -( R ) chains, cyclic ( R ) oligomers, oligolides, and polymers composed of rac -hydroxybutanoate, HB, but not all -( S ) or syndiotactic ( R, S ) chains. We have now studied the ability for configurational recognition (stereoselectivity) by the purified PHB depolymerase from Alcaligenes faecalis T 1 . To this end, a titristat/HPLC method has been developed for following the degradation of oligo(3-hydroxybutanoates), OHBs, providing baseline separation for OHB methyl esters up to at least the octamers (Figure 1). With this method, we have now investigated the degradation rates and cleavage patterns of OHBs ( 1 − 16 ) containing up to eight HB units with given sequences of ( R ) and ( S ) configurations along the chains (Figures 2−5 and 7). Analysis of the measurements now allows us to propose more detailed structural features at the binding site of the depolymerase (Figures 6, 8, 9): (i) the enzyme is an endo esterase; (ii) it recognizes the orientation of the chain relative to its active site; (iii) the binding site contains four subsites, three of which have to be occupied by HB units for cleavage to occur at all (rate v max β) and all four for cleavage to take place at the maximum rate ( v max α); and (iv) the central two subsites, between which cleavage occurs, must be occupied by ( R ) HB units, whereas the terminal subunits may also be occupied by ( S ) HB units.