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The early-lineage, aerobic, zoosporic fungi from the Chytridiomycota constitute less than 1% of the described fungi and can use diverse sources of nutrition from plant or animal products. One of the ancestral sources of fungal nutrition could be products following enzymatic degradation of plant material. However, carbohydrate-active enzymes from these ancient fungi have been less studied. A GH11 xylanase (RrXyn11A) (EC 3.2.1.8) and a GH43 xylosidase (RrXyl43A) (EC 3.2.1.37) were identified from an early-lineage aerobic zoosporic fungus, Rhizophlyctis rosea NBRC 105426. Both genes were heterologously expressed in Pichia pastoris and the recombinant enzymes were purified and characterized. The optimal pH for recombinant RrXyn11A and RrXyl43A was pH 7. RrXyn11A had high stability over a wide range of pH (4-8) and temperature (25-70 °C). RrXyn11A also showed high substrate specificity on both azurine-cross-linked (AZCL) arabinoxylan and AZCL xylan. RrXyl43A had β-xylosidase and minor α-L-arabinofuranosidase activity. This enzyme showed low product inhibition and retained 51% activity in the presence of 100 mM xylose. A combination of RrXyn11A and RrXyl43A exhibited significantly higher hydrolytic and polymer degradation capability and xylose release on wheat bran and beechwood xylan compared to treatment with commercial enzymes. This study was the first to heterologously express and characterize the GH11 xylanase (RrXyn11A) and GH43 xylosidase (RrXyl43A) from the ancient fungus, R. rosea. Meanwhile, this study also demonstrated that the enzymes from the ancient fungus R. rosea can be easily handled and heterologously expressed in Pichia, which presents a promising path to a new source of enzymes for biomass degradation. [ABSTRACT FROM AUTHOR]

Type A chitinases (EC 3.2.1.14), GH family 18, attack chitin ((1 → 4)-2-acetamido-2-deoxy-β- d-glucan) and chito-oligosaccharides from the reducing end to catalyze release of chitobiose ( N, N′-diacetylchitobiose) via hydrolytic cleavage of N-acetyl-β- d-glucosaminide (1 → 4)-β-linkages and are thus ' exo-chitobiose hydrolases.' In this study, the chitinase type A from Serratia marcescens (SmaChiA) was used as a template for identifying two novel exo-chitobiose hydrolase type A enzymes, FbalChi18A and MvarChi18A, originating from the marine organisms Ferrimonas balearica and Microbulbifer variabilis, respectively. Both FbalChi18A and MvarChi18A were recombinantly expressed in Escherichia coli and were confirmed to exert exo-chitobiose hydrolase activity on chito-oligosaccharides, but differed in temperature and pH activity response profiles. Amino acid sequence comparison of the catalytic β/α barrel domain of each of the new enzymes showed individual differences, but ~69% identity of each to that of SmaChiA and highly conserved active site residues. Superposition of a model substrate on 3D structural models of the catalytic domain of the enzymes corroborated exo-chitobiose hydrolase type A activity for FbalChi18A and MvarChi18A, i.e., substrate attack from the reducing end. A main feature of both of the new enzymes was the presence of C-terminal 5/12 type carbohydrate-binding modules (SmaChiA has no C-terminal carbohydrate binding module). These new enzymes may be useful tools for utilization of chitin as an N-acetylglucosamine donor substrate via chitobiose. [ABSTRACT FROM AUTHOR]

Fungal laccases (EC 1.10.3.2) are multi-copper oxidases that oxidize a wide variety of substrates. Despite extensive studies, the molecular basis for their diverse activity is unclear. Notably, there is no current way to rationally predict the activity of a laccase toward a given substrate. Such knowledge would greatly facilitate the rational design of new laccases for technological purposes. We report a study of three datasets of experimental Km values and activities for Trametes versicolor and Cerrena unicolor laccase, using a range of protein modeling techniques. We identify diverse binding modes of the various substrates and confirm an important role of Asp-206 and His-458 (T. versicolor laccase numbering) in guiding substrate recognition. Importantly, we demonstrate that experimental Km values correlate with binding affinities computed by MMGBSA. This confirms the common assumption that the protein-substrate affinity is a major contributor to observed Km. From quantitative structure-activity relations (QSAR) we identify physicochemical properties that correlate with observed Km and activities. In particular, the ionization potential, shape, and binding affinity of the substrate largely determine the enzyme’s Km for the particular substrate. Our results suggest that Km is not just a binding constant but also contains features of the enzymatic activity. In addition, we identify QSAR models with only a few descriptors showing that phenolic substrates employ optimal hydrophobic packing to reach the T1 site, but then require additional electronic properties to engage in the subsequent electron transfer. Our results advance our ability to model laccase activity and lend promise to future rational optimization of laccases toward phenolic substrates. [ABSTRACT FROM AUTHOR]