AbstractPolyhydroxyalkanoate synthase, PhaC, is a key enzyme in the biosynthesis of PHA, a type of bioplastics with huge potential to replace conventional petroleum-based plastics. While two PhaC structures have been determined recently, the exact mechanism remains unclear partly due to the absence of a tunnel for product passage. The PhaC fromAquitaleasp. USM4, PhaCAq, was characterised and showed aKmof 394 µM and akcatof 476.4 s−1on the 3HB-CoA substrate. A model based on the structure of the closely related PhaC fromCupriavidus necator, PhaCCnrevealed a three-branched tunnel at the dimeric interface. Two of the branches open to the solvent and serve as the putative routes for substrate entrance and product exit, while the third branch is elongated in a PhaC1 model fromPseudomonas aeruginosa, indicating a function of accommodating the hydroxyalkanoate (HA) moiety of the HA-CoA substrate. Docking of the two tetrahedral intermediates formed during catalysis suggests a PHA elongation mechanism that requires the HA moiety of the ligand to rotate ~180°. Both classes I and II PhaCs share a common mechanism for polymer elongation, and substrate specificity is determined in part by a bulky Phe/Tyr/Trp residue in the third branch in class I, which is conserved as Ala in class II to create room for longer substrates. The PhaCAqmodel provides fresh insights into a general PhaC mechanism, pinpointing key residues for potential engineering of PhaCs with desirable characteristics.
A Substrate Specificity-determining Unit of Three Lin12-Notch Repeat Modules Is Formed inTranswithin the Pappalysin-1 Dimer and Requires a Sequence Stretch C-terminal to the Third Module
