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 Alcaligenes faecalis 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 vmaxβ) and all four for cleavage to take place at the maximum rate (vmaxR); 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.

A New Type of Thermoalkalophilic Hydrolase of Paucimonas lemoignei with High Specificity for Amorphous Polyesters of Short Chain-length Hydroxyalkanoic Acids

“…This result confirmed that the purified depolymerase has endo-hydrolase activity (see above, results with aPHB). Oligomers of (S)-3HB were not hydrolyzed at all and confirmed the specificity of the enzyme for the R isomers as it has been found for dPHB depolymerase of Alcaligenes faecalis (13).…”

“…However, the enzyme hydrolyzed aPHB when this amorphous polymer was blended, or copolymerized, with a variety of crystalline polyesters (10 -12). Hydrolytic activity toward aPHB was clearly of an endo type because of the presence of S units along the chain, which are known not to be recognized by PHB depolymerases (10,13). The conclusion was drawn that in aPHB, efficient enzyme binding to the substrate is prevented by exceeding mobility of the polymer chains in the amorphous rubbery state.…”

The “Intracellular” Poly(3-Hydroxybutyrate) (PHB) Depolymerase of Rhodospirillum rubrum Is a Periplasm-Located Protein with Specificity for Native PHB and with Structural Similarity to Extracellular PHB Depolymerases

“…For determination of temperature stability, purified PhaZ1 (0.6 g) was incubated in assay buffer at 50°C for the times indicated before residual activity was determined (columns). 2 or MgCl 2 increased the activity to 130 or 140% at 1 mM, respectively, but decreased the activity at 5 mM (both to 75%) or at 10 mM (to 40 or 35%, respectively). EDTA partially inhibited PhaZ1, but a 5 mM concentration was necessary to obtain more than 50% inhibition.…”

Enzymes catalysing the synthesis and degradation of beta-linked biopolymers and their applications

“…Bachmann and Seebach proposed that PhaZ RpiT1 has four subsites (2, 1, -1, and -2) in its active site, in which three of the subsites must be occupied by 3HB units for cleavage to occur at the centre of the active site [106]. Homology modelling of PhaZ RpiT1 (the modelled residue range was from positions 124 to 294) based on the crystal structure of PhaZ Pfu suggested that Asn285 (color-coded according to molecular species) of PhaZ RpiT1 is located at the mouth of the crevice, immediately above His273, which probably involved subsite -1 (Figure 7 (A)).…”