Revealing the essential role of the lid in mclPHA intracellular depolymerase from Pseudomonas putida KT2440

Abstract Polyhydroxyalkanoates (PHAs) are microbial polyesters that serve as intracellular carbon reserves and represent promising biodegradable alternatives to conventional plastics. However, their large-scale application requires not only cost-effective production but also efficient strategies for recovery and recycling. Unlike short-chain-length PHAs, which are widely degraded by diverse enzymes, the intracellular degradation of medium-chain-length PHAs (mclPHAs) appears to be a genus-specific trait of  Pseudomonas . In this context, the PhaZKT depolymerase from Pseudomonas putida KT2440 is considered a model enzyme for intracellular mclPHA mobilization; it is highly substrate-specific, acting almost exclusively on mclPHAs, and consists of an α/β-hydrolase fold with a lid domain, similar to lipases and other enzymes acting on lipid substrates, in contrast to extracellular PHA depolymerases, which generally lack this lid structure. Here, we explored the essential role of this lid structure through site-directed deletions and random mutagenesis. Targeted deletions within or near the lid completely abolished enzyme activity, highlighting its critical structural and functional importance. Random mutagenesis identified two beneficial variants: S184F, located in the lid hinge region, and G286R, situated in a still unmapped region. The S184F mutant exhibited increased esterase activity on p -nitrophenyl esters but significantly reduced depolymerase activity on mclPHA nanoparticles, indicating that lid integrity and dynamics precisely control substrate specificity and access. Molecular dynamics simulations supported these findings, revealing enhanced rigidity near the lid region in the S184F variant. Conversely, G286R showed substantially improved depolymerase activity toward mclPHA, suggesting alternative regions for beneficial mutations without compromising lid functionality. These results underscore the delicate balance between lid integrity and enzyme performance, offering insights into targeted protein engineering for optimized enzymatic recycling of bioplastics. Key points •  The lid in PhaZKT is essential for depolymerase activity •  All lid-targeted mutants completely lost enzymatic activity •  Random mutagenesis identified two active distal mutants