Background:The recently discovered lytic polysaccharide monooxygenases (LPMOs) are important in enzymatic conversion of lignocellulosic biomass. Results: We describe structural and functional studies of NcLPMO9C, which cleaves both cellulose and certain hemicelluloses. Conclusion: NcLPMO9C has structural and functional features that correlate with the enzyme's catalytic capabilities. Significance: This study shows how LPMO active sites are tailored to varying functionalities and adds to a growing LPMO knowledge base.
Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds using molecular oxygen and an external electron donor. We have used NMR and isothermal titration calorimetry (ITC) to study the interactions of a broad-specificity fungal LPMO, NcLPMO9C, with various substrates and with cellobiose dehydrogenase (CDH), a known natural supplier of electrons. The NMR studies revealed interactions with cellohexaose that center around the copper site. NMR studies with xyloglucans, i.e., branched β-glucans, showed an extended binding surface compared with cellohexaose, whereas ITC experiments showed slightly higher affinity and a different thermodynamic signature of binding. The ITC data also showed that although the copper ion alone hardly contributes to affinity, substrate binding is enhanced for metal-loaded enzymes that are supplied with cyanide, a mimic of O 2 − . Studies with CDH and its isolated heme b cytochrome domain unambiguously showed that the cytochrome domain of CDH interacts with the copper site of the LPMO and that substrate binding precludes interaction with CDH. Apart from providing insights into enzyme-substrate interactions in LPMOs, the present observations shed new light on possible mechanisms for electron supply during LPMO action.
Cutinases are α/β hydrolases, and their role in nature is the degradation of cutin. Such enzymes are usually produced by phytopathogenic microorganisms in order to penetrate their hosts. The first focused studies on cutinases started around 50 years ago. Since then, numerous cutinases have been isolated and characterized, aiming at the elucidation of their structure–function relations. Our deeper understanding of cutinases determines the applications by which they could be utilized; from food processing and detergents, to ester synthesis and polymerizations. However, cutinases are mainly efficient in the degradation of polyesters, a natural function. Therefore, these enzymes have been successfully applied for the biodegradation of plastics, as well as for the delicate superficial hydrolysis of polymeric materials prior to their functionalization. Even though research on this family of enzymes essentially began five decades ago, they are still involved in many reports; novel enzymes are being discovered, and new fields of applications arise, leading to numerous related publications per year. Perhaps the future of cutinases lies in their evolved descendants, such as polyesterases, and particularly PETases. The present article reviews the biochemical and structural characteristics of cutinases and cutinase-like hydrolases, and their applications in the field of bioremediation and biocatalysis.
The increasing demand for the development of efficient biocatalysts is a consequence of their broad industrial applications. Typical difficulties that are encountered during their exploitation in a variety of processes are interconnected with factors such as temperature, pH, product inhibitors etc. To eliminate these, research has been directed towards the identification of new enzymes that would comply with the required standards. To this end, the recently discovered glucuronoyl esterases (GEs) are an enigmatic family within the carbohydrate esterase (CE) family. Structures of the thermophilic StGE2 esterase from Myceliophthora thermophila (synonym Sporotrichum thermophile), a member of the CE15 family, and its S213A mutant were determined at 1.55 and 1.9 Å resolution, respectively. The first crystal structure of the S213A mutant in complex with a substrate analogue, methyl 4-O-methyl-β-D-glucopyranuronate, was determined at 2.35 Å resolution. All of the three-dimensional protein structures have an α/β-hydrolase fold with a three-layer αβα-sandwich architecture and a Rossmann topology and comprise one molecule per asymmetric unit. These are the first crystal structures of a thermophilic GE both in an unliganded form and bound to a substrate analogue, thus unravelling the organization of the catalytic triad residues and their neighbours lining the active site. The knowledge derived offers novel insights into the key structural elements that drive the hydrolysis of glucuronic acid esters.
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