Glycoside hydrolase family 31 (GH31) enzymes show both highly conserved folds and catalytic residues. Yet different members of GH31 show very different substrate specificities, and it is not obvious how these specificities arise from the protein sequences. The fungal α-xylosidase, AxlA, was originally isolated from a commercial enzyme mixture secreted by Aspergillus niger and was reported to have potential as a catalytic component in biomass deconstruction in the biofuel industry. We report here the crystal structure of AxlA in complex with its catalytic product, a hydrolyzed xyloglucan oligosaccharide. On the basis of our new structure, we provide the structural basis for AxlA’s role in xyloglucan utilization and, more importantly, a new procedure to predict and differentiate C5 vs C6 sugar specific activities based on protein sequences of the functionally diverse GH31 family enzymes.

The functional and biological significance of selected CASP13 targets are described by the authors of the structures. The structural biologists discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP13 experiment.

This book covers the history, chemistry, and biochemistry of cyclic peptide toxins of Amanita and other poisonous mushrooms.

Cyclic peptides are promising compounds for new chemical biological tools and therapeutics due to their structural diversity, resistance to proteases, and membrane permeability. Amatoxins, the toxic principles of poisonous mushrooms, are biosynthesized on ribosomes as 35mer precursor peptides, which are ultimately converted to hydroxylated bicyclic octapeptides. The initial cyclization steps, catalyzed by a dedicated prolyl oligopeptidase (POPB), involves removal of the 10-amino acid leader sequence from the precursor peptide and transpeptidation to produce a monocyclic octapeptide intermediate. The utility of POPB as a general catalyst for peptide cyclization was systematically characterized using a range of precursor peptide substrates produced either in E. coli or chemically. Substrates produced in E. coli were expressed either individually or in mixtures produced by codon mutagenesis. A total of 127 novel peptide substrates were tested, of which POPB could cyclize 100. Peptides of 7-16 residues were cyclized at least partially. Synthetic 25mer precursor peptide substrates containing modified amino acids including d-Ala, β-Ala, N-methyl-Ala, and 4-hydroxy-Pro were also successfully cyclized. Although a phalloidin heptapeptide with all L amino acids was not cyclized, partial cyclization was seen when l-Thr at position #5 was replaced with the naturally occurring D amino acid. POPB should have broad applicability as a general catalyst for macrocyclization of peptides containing 7 to at least 16 amino acids, with an optimum of 8-9 residues.

Bananas and plantains (Musa spp.) are among the most important crops in the world providing staple food for hundreds of millions of people. However, banana production has been devastated by fungal infestations caused by Fusarium oxysporum f. sp. cubense (Foc). Despite the fact that there is very little known on the role of microbial metabolites in the molecular mechanism of Foc infections, it has been proposed that the toxins fusaric acid and beauvericin produced by Foc play an important role during pathogenesis. The aim of this contribution was to study the toxic components of culture filtrates (CF) of Foc and to isolate the extracellular microbial metabolites involved in the plant response. An in vitro bioassay was used to evaluate the production of phytotoxic metabolites as well as the specificity of culture from a strain of Foc belonging to VCG 01210 (race 1). A host‐specific CF was obtained and the phytotoxic compounds characterized as fusaric acid, beauvericin and fumonisin B1. Despite the presence of these nonspecific toxins, a water‐soluble extract from the CF induced protection to the main phytotoxic fraction, measured by lesion area. This hydrophilic fraction induced a fast and strong response of just jasmonic acid (JA)‐dependent defence genes rather than salicylic acid (SA)‐ and ethylene (ET)‐response genes in resistant cultivars. Extracellular proteins isolated from CF of Foc provide an important source for further investigations on the molecular basis of the interaction between Foc and banana.

Lignin content and composition are crucial factors affecting biomass digestibility. Exploring the genetic loci simultaneously affecting lignin-relevant traits and biomass digestibility is a precondition for lignin genetic manipulation towards energy crop breeding. In this study, a high-throughput platform was employed to assay the lignin content, lignin composition and biomass enzymatic digestibility of a rice recombinant inbred line population. Correlation analysis indicated that the absolute content of lignin monomers rather than lignin content had negative effects on biomass saccharification, whereas the relative content of p -hydroxyphenyl unit and the molar ratio of p -hydroxyphenyl unit to guaiacyl unit exhibited positive roles. Eight QTL clusters were identified and four of them affecting both lignin composition and biomass digestibility. The additive effects of clustered QTL revealed consistent relationships between lignin-relevant traits and biomass digestibility. Pyramiding rice lines containing the above four positive alleles for increasing biomass digestibility were selected and showed comparable lignin content, decreased syringyl or guaiacyl unit and increased molar percentage of p -hydroxyphenyl unit, the molar ratio of p -hydroxyphenyl unit to guaiacyl unit and sugar releases. More importantly, the lodging resistance and eating/cooking quality of pyramiding lines were not sacrificed, indicating the QTL information could be applied to select desirable energy rice lines.

The high cost of enzymes is one of the key technical barriers that must be overcome to realize the economical production of biofuels and biomaterials from biomass. Supplementation of enzyme cocktails with lytic polysaccharide monooxygenase (LPMO) can increase the efficiency of these cellulase mixtures for biomass conversion. The previous studies have revealed that LPMOs cleave polysaccharide chains by oxidization of the C1 and/or C4 carbons of the monomeric units. However, how LPMOs enhance enzymatic degradation of lignocellulose is still poorly understood.

In Trichoderma reesei light is an important factor in the regulation of glycoside hydrolase gene expression. We therefore investigated the influence of different light intensities on cellulase activity and protein secretion. Differentially secreted proteins in light and darkness as identified by mass spectrometry included members of different glycoside hydrolase families, such as CBH1, Cel3A, Cel61B, XYN2, and XYN4. Several of the associated genes showed light-dependent regulation on the transcript level. Deletion of the photoreceptor genes blr1 and blr2 resulted in a diminished difference of protein abundance between light and darkness. The amount of secreted proteins including that of the major exo-acting beta-1,4-glucanases CBH1 and CBH2 was generally lower in light-grown cultures than in darkness. In contrast, cbh1 transcript levels increased with increasing light intensity from 700 to 2,000 lux but dopped at high light intensity (5,000 lux). In the photoreceptor mutants Δblr1 and Δblr2 cellulase activity in light was reduced compared to activity in darkness, showing a discrepancy between transcript levels and secreted cellulase activity. Furthermore, evaluation of different light sensitivities revealed an increased light tolerance with respect to cellulase expression of QM9414 compared to its parental strain QM6a. Investigation of one of the differentially expressed proteins between light and darkness, CLF1, revealed its function as a factor involved in regulation of secreted protease activity. T. reesei secretes a different set of proteins in light compared to darkness, this difference being mainly due to the function of the major known photoreceptors. Moreover, cellulase regulation is adjusted to light intensity and improved light tolerance was correlated with increased cellulase production. Our findings further support the hypothesis of a light intensity dependent post-transcriptional regulation of cellulase gene expression in T. reesei.

Endo-β1,4-glucanases in glycosyl hydrolase family 5 (GH5) are ubiquitous enzymes in the multicellular fungi and are common components of enzyme cocktails for biomass conversion. We recently showed that an endo-glucanase of subfamily 5 of GH5 (GH5_5) from Sporotrichum thermophile (StCel5A) was more effective at releasing glucose from pretreated corn stover, when part of an eight-component synthetic enzyme mixture, compared to its closely related counterpart from Trichoderma reesei, TrCel5A. StCel5A and TrCel5A belong to different clades of GH5_5 (GH5_5_1 and GH5_5_2, respectively). To test whether the superior activity of StCel5A was a general property of all enzymes in the GH5_5_2 clade, StCel5A, TrCel5A, and two additional members of each subfamily were expressed in a common host that had been engineered to suppress its native cellulases (T. reesei Δxyr1) and compared against each other alone on pure substrates, in synthetic mixtures on pure substrates, and against each other in synthetic mixtures on real biomass. The results indicated that superiority is a unique property of StCel5A and not of GH5_5_2 generally. The six Cel5A enzymes had significant differences in relative activities on different substrates, in specific activities, and in sensitivities to mannan inhibition. Importantly, the behavior of the six endo-glucanases on pure cellulose substrates did not predict their behavior in combination with other cellulolytic enzymes on a real lignocellulosic biomass substrate.

Many host genes induced during compatible plant-pathogen interactions constitute targets of pathogen virulence factors that act to suppress host defenses. In order to identify Nicotiana tabacum L. genes for pathogen-induced proteins involved in susceptibility to the oomycete Phytophthora parasitica var. nicotianae, we used SuperSAGE technology combined with next-generation sequencing to identify transcripts that were differentially upregulated during a compatible interaction. We identified a pathogen-induced gene (NtPIP) that was rapidly induced only during the compatible interaction. Virus-induced gene silencing of NtPIP reduced the susceptibility of N. tabacum to P. parasitica var. nicotianae. Additionally, transient expression of NtPIP in the resistant species Nicotiana megalosiphon Van Heurck & Mull. Arg. compromised the resistance to P. parasitica var. nicotianae. This pathogen-induced protein is therefore a positive regulator of the susceptibility response against an oomycete pathogen in tobacco.