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Abstract
Polyhydroxyalkanoate (PHA) is a promising candidate for use as an alternative bioplastic to replace petroleum-based plastics. Our understanding of PHA synthase PhaC is poor due to the paucity of available three-dimensional structural information. Here we present a high-resolution crystal structure of the catalytic domain of PhaC from Chromobacterium sp. USM2, PhaCCs-CAT. The structure shows that PhaCCs-CAT forms an α/β hydrolase fold comprising α/β core and CAP subdomains. The active site containing Cys291, Asp447 and His477 is located at the bottom of the cavity, which is filled with water molecules and is covered by the partly disordered CAP subdomain. We designated our structure as the closed form, which is distinct from the recently reported catalytic domain from Cupriavidus necator (PhaCCn-CAT). Structural comparison showed PhaCCn-CAT adopting a partially open form maintaining a narrow substrate access channel to the active site, but no product egress. PhaCCs-CAT forms a face-to-face dimer mediated by the CAP subdomains. This arrangement of the dimer is also distinct from that of the PhaCCn-CAT dimer. These findings suggest that the CAP subdomain should undergo a conformational change during catalytic activity that involves rearrangement of the dimer to facilitate substrate entry and product formation and egress from the active site.
Highlights
PHA synthase (PhaC) is the key enzyme involved in PHA biosynthesis and functions by polymerizing monomeric hydroxyalkanoate substrates[3, 13, 14]
We have examined the structural details in an effort to arrive at a possible mechanism by which the conformational difference between PhaCCs-CAT and PhaC from Cupriavidus necator/Ralstonia eutropha (PhaCCn)-CAT is induced at the CAP subdomains, and found that one disulfide bond is formed between Cys[382] and Cys[438] in the PhaCCn-CAT structures[22, 23] (Fig. 4c)
Inspection of the PhaCCs-CAT and PhaCCn-CAT structures suggests that the catalytic domain of PHA synthases exists in an open-closed equilibrium in solution by means of conformational changes in the CAP subdomain (Fig. 7a,b)
Summary
PHA synthase (PhaC) is the key enzyme involved in PHA biosynthesis and functions by polymerizing monomeric hydroxyalkanoate substrates[3, 13, 14]. In the course of our structural studies, two independent structural investigations of the catalytic domain of PhaC from Cupriavidus necator (the old code is Ralstonia eutropha), PhaCCn-CAT (both at 1.8 Å), have been reported[22, 23]. These studies have essentially reported the same structure comprising a partially open form, the proposed enzyme mechanisms differ. Our three-dimensional structure of PhaCCs together with PhaCCn provides valuable information concerning the enzyme dynamics and should assist in delineation of the enzymatic mechanisms utilized by the important and related synthases, in addition to providing the basis for facilitating structure-based improvement of the enzymatic activity
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