Structural and Biophysical Analysis of the Phytochelatin Synthase Like Enzyme from Nostoc sp. Shows That Its Protease Activity is Sensitive to the Redox State of the Substrate

Phytochelatins PCs are nonribosomal thiol rich oligopeptides synthetized from glutathione GSH in a amp; 947; glutamylcysteinyl transpeptidation reaction catalyzed by PC synthases PCSs . Ubiquitous in plant and present in some invertebrates, PCSs are involved in metal detoxification and homeostasis. The PCS like enzyme from the cyanobacterium Nostoc sp. NsPCS is considered to be an evolutionary precursor enzyme of genuine PCSs because it shows sufficient sequence similarity for homology to the catalytic domain of the eukaryotic PCSs and shares the peptidase activity consisting in the deglycination of GSH. In this work, we investigate the catalytic mechanism of NsPCS by combining structural, spectroscopic, thermodynamic, and theoretical techniques. We report several crystal structures of NsPCS capturing different states of the catalyzed chemical reaction i the structure of the wild type enzyme wt NsPCS ; ii the high resolution structure of the amp; 947; glutamyl cysteine acyl enzyme intermediate acyl NsPCS ; and iii the structure of an inactive variant of NsPCS, with the catalytic cysteine mutated into serine C70S NsPCS . We characterize NsPCS as a relatively slow enzyme whose activity is sensitive to the redox state of the substrate. Namely, NsPCS is active with reduced glutathione GSH , but is inhibited by oxidized glutathione GSSG because the cleavage product is not released from the enzyme. Our biophysical analysis led us to suggest that the biological function of NsPCS is being a part of a redox sensing system. In addition, we propose a mechanism how PCS like enzymes may have evolved toward genuine PCS enzyme

Investigation of the halophilic PET hydrolase PET6 from Vibrio gazogenes

The handling of plastic waste and the associated ubiquitous occurrence of microplastic poses one of the biggest challenges of our time. Recent investigations of plastic degrading enzymes have opened new prospects for biological microplastic decomposition as well as recycling applications. For polyethylene terephthalate, in particular, several natural and engineered enzymes are known to have such promising properties. From a previous study that identified new PETase candidates by homology search, we chose the candidate PET6 from the globally distributed, halophilic organism Vibrio gazogenes for further investigation. By mapping the occurrence of Vibrios containing PET6 homologs we demonstrated their ubiquitous prevalence in the pangenome of several Vibrio strains. The biochemical characterization of PET6 showed that PET6 has a comparatively lower activity than other enzymes but also revealed a superior turnover at very high salt concentrations. The crystal structure of PET6 provides structural insights into this adaptation to saline environments. By grafting only a few beneficial mutations from other PET degrading enzymes onto PET6, we increased the activity up to three fold, demonstrating the evolutionary potential of the enzyme. MD simulations of the variant helped rationalize the mutational effects of those mutants and elucidate the interaction of the enzyme with a PET substrate. With tremendous amounts of plastic waste in the Ocean and the prevalence of Vibrio gazogenes in marine biofilms and estuarine marshes, our findings suggest that Vibrio and the PET6 enzyme are worthy subjects to study the PET degradation in marine environment