Fungal Counterparts Research Articles

Biochar as a novel niche for culturing microbial communities in composting

Biochar has been applied as a bulk agent or an additive to compost. The mixture of biochar and compost has been considered to exert synergistic effect as a soil amendment. In a composting system, the macro-porous sites of biochar may act as a novel niche that selects and cultures the microorganisms from the bulk compost. A variety of volatile organic carbons (VOCs) such as aromatic hydrocarbons and aliphatics were detected in biochar pellets (BC) pyrolyzed at 100°C. In the mesosphilic phase, the water-soluble carbon (WSC) and water-soluble phenols (WSP) in biochar increased from 2.1 to 26mgkg−1 and 5.9 to 101μgkg−1, respectively. These labile carbons however, were subjected to a rapid metabolism over the composting course. We further compared the responses of microbial community in BC to those in the bulk organic matter. Both Shannon-Wiener and Richness indexes of bacterial communities were higher in BC than in the adjacent compost (ADJ) and the bulk organic matter (control). As for fungal communities, the two indexes were higher in BC than ADJ and control only in the mature phase. During the composting course, the bacterial activity was higher than the fungal counterpart in terms of the changes of corresponding biomarkers, glucosamine and muramic acids. The results suggested that the diversified labile carbons sources including VOCs and WSC in BC could influence the structure of microbial community and resulted in an enhanced carbon catabolic capacity.

Structural and inhibitor screening studies of a fungal form of aspartate semialdehyde dehydrogenase from Cryptococcus neoformans

Aspartate semialdehyde dehydrogenase (ASADH) functions at a critical junction in the aspartate biosynthetic pathway and represents a validated target for antimicrobial drug design. This enzyme catalyzes the NADPH‐dependent reductive dephosphorylation of β‐aspartyl phosphate to produce the key intermediate aspartate semialdehyde. Production of this intermediate represents the first committed step for the biosynthesis of essential amino acids in fungi and in bacteria. The absence of this enzyme in humans and other mammals will allow selective targeting of pathogenic microorganisms. We have accumulated significant structural and mechanistic information about the bacterial ASADHs, but have only limited knowledge of their fungal counterparts. The structure of a new fungal form of ASADH from Cryptococcus neoformans has been determined to 2.6 Å resolution. The overall structure of CnASADH is similar to that of its bacterial orthologs, but with some critical differences both in biological assembly and in secondary structural features that can potentially be exploited for the development of species selective drugs with selective toxicity against only certain infectious organisms. A customized fragment library has been screened against several fungal ASADHs and initial hits have been identified with inhibition constants (Ki) in the low millimolar range. The ASADH obtained from this fungal species have shown different inhibition selectivity when compared to this enzyme from bacterial species and also differences between fungal species. Docking studies with the CnASADH structure are being used to guide the design and development of potent inhibitors. The long term goal of this project is to develop drugs that selectively target this enzyme and block the aspartate biosynthetic pathway.Support or Funding InformationThis work is supported by funding from the NIH (AI077720).

Microbial community structure and predicted bacterial metabolic functions in biochar pellets aged in soil after 34 months

Biochar has been shown to induce changes in soil microbial communities, which is initially attributed to the labile organic carbons stimulating degraders over short time periods. However, little is currently understood about microbial communities as well as their metabolic traits on biochar pellets (BC) aged in soil for prolonged periods of time. It was hypothesized that microbial communities affected by biochar may result in changes in the metabolic traits. The activities of bacteria and fungi were compared by measuring their biomarkers including muramic acid (MurN) and glucosamine (GluN). The structure of soil microbial communities on BC aged in soil after 34 months was evaluated using 454 pyrosequencing. The metabolic functions of the bacterial community were predicted by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). Pyrolysates in BC were analyzed using pyrolysis-gas chromatography/mass spectroscopy. The statistically significant decrease of GluN/MurN in BC in comparison with the adjacent soil (ADJ) and the control indicates higher accumulated bacterial activity than that of fungi. The structure of bacterial communities greatly shifted in biochar pellets in comparison with their fungal counterparts. Proteobacteria and Actinobacteria of bacterial community were more abundant in BC than in ADJ and control soils. At class level there were greater bacterial OTUs in BC than ADJ and control soils. The changes of pyrolysates in BC may indicate the deconstruction of biochar. The PICRUSt indicated the bacterial community in BC shifted from preferring metabolizing carbohydrates to xenobiotics. This was supported by the high concentration of water soluble carbon present in BC. The current study suggests that biochar may preserve soil organic matter through mediating bacterial metabolic capacities in carbon sources.

Endo-glucanase digestion of oat β-Glucan enhances Dectin-1 activation in human dendritic cells

Oat β-Glucans were studied for their immunological impact before and after enzymatic digestion in order to enhance the efficacy of oat β-Glucans for application in functional foods. Oat β-Glucan is reported to have minimal impact compared to its fungal counterpart in vitro. Digestion with endo-glucanase enhanced its efficacy towards stimulating MCP-1, RANTES, IL-8, and IL-4 production in human dendritic cells as compared to the nondigested β-Glucan. This effect resulted from an enhanced activation of the Dectin-1 receptor. Our data suggest that the immune-stimulation was dependent on the β-(1-3) linkages and the reduced particle size of digested β-Glucans. Thus, we show that enzymatic pre-digestion of dietary fibres such as oat β-Glucan enhances its impact on specific immune receptors. We also demonstrate that particle size and/or molecular weight of oat β-Glucans and exposure of specific binding sites for the receptors might be important tools for designing efficacious functional feed and food additives.

KRAFT LIGNIN DEGRADATION BY AUTOCHTONOUS STREPTOMYCES STRAINS ISOLATED FROM A TROPICAL LAGOON ECOSYSTEM

Kraft lignin contributes to the toxicity of the pulping plant effluent and is known to resist microbial treatment.The lignin component must be removed from lignocellulose biomass to enhance the release of fermentable sugars for the production of biofuel and other value-added end products. Lignin-degrading bacteria provide an advantage due to their ease of isolation,wider tolerance of environmental conditions and genetic manipulations compared with their fungal counterparts. There is no documented evidence on the degradation of kraft lignin by bacteria in the tropical estuarine ecological niche in Nigeria. Bacterial growth and assessment of kraft-lignin degradation in submerged fermentation was carried out for a period of 10 days using Streptomyces spp isolated from a tropical lagoon as the inocula. The organisms utilized 23 to 99 % kraft-lignin at the rate of 2.3×10-5 to 9.9×10-5 g.d-1cm-3 with specific growth rates of 0.020 - 0.084 h-1and doubling times of 8.3 - 35.1 h. Maximum values obtained for laccase and peroxidase activities were 9.5x10-2 and 400 μ mol mg -1min -1 respectively. The aim of this study was to obtain evidences for Kraft lignin degradation by indigenous tropical estuarine Streptomyces species from Lagos, Nigeria. The Autochthonous bacterial species of the Lagos lagoon utilize kraft lignin as a sole carbon source and may be good candidates for biotechnological purposes. The outcome of this study has bridged an information gap in the tropical environment and will complement existing global data because the information on the degradation of kraft lignin by marine Streptomyces is not common.

Specific Activation of the Plant P-type Plasma Membrane H+-ATPase by Lysophospholipids Depends on the Autoinhibitory N- and C-terminal Domains

Eukaryotic P-type plasma membrane H(+)-ATPases are primary active transport systems that are regulated at the post-translation level by cis-acting autoinhibitory domains, which can be relieved by protein kinase-mediated phosphorylation or binding of specific lipid species. Here we show that lysophospholipids specifically activate a plant plasma membrane H(+)-ATPase (Arabidopsis thaliana AHA2) by a mechanism that involves both cytoplasmic terminal domains of AHA2, whereas they have no effect on the fungal counterpart (Saccharomyces cerevisiae Pma1p). The activation was dependent on the glycerol backbone of the lysophospholipid and increased with acyl chain length, whereas the headgroup had little effect on activation. Activation of the plant pump by lysophospholipids did not involve the penultimate residue, Thr-947, which is known to be phosphorylated as part of a binding site for activating 14-3-3 protein, but was critically dependent on a single autoinhibitory residue (Leu-919) upstream of the C-terminal cytoplasmic domain in AHA2. A corresponding residue is absent in the fungal counterpart. These data indicate that plant plasma membrane H(+)-ATPases evolved as specific receptors for lysophospholipids and support the hypothesis that lysophospholipids are important plant signaling molecules.

Insights into the substrate specificity and synergy with mannanase of family 27 α-galactosidases from Neosartorya fischeri P1.

Thermophilic Neosartorya fischeri P1 is an excellent carbohydrate-active enzyme (CAZyme) producer. Two α-galactosidases of GH (glycoside hydrolase ) family 27 with a very low sequence identity (28.7%), Gal27A and Gal27B, were identified in strain P1 and functionally expressed in Pichia pastoris. In comparison to other characterized GH27 fungal counterparts, rGal27B has a higher temperature optimum (75 °C) and better thermostability (>50% activity at 70 °C for 15 min), and rGal27A shows stability over the broadest pH range (pH 2.0-12.0). Moreover, great distinctions lie in the two enzymes. When using pNPG as the substrate, rGal27B had a higher turnover number (1621.4 vs. 368.3 s(-1)) but lower affinity (2.84 vs. 0.8 mM) and catalytic efficiency (460.8 vs. 580.3 s(-1) mM(-1)) than rGal27A. rGal27B acted on galacto-oligosaccharides, whereas rGal27A was active on polymeric substrates. Although both enzymes showed synergy in galactomannan degradation when combined with a β-mannanase of the same strain, enzyme combinations including rGal27A released more reducing sugars (up to 11.67-fold). Homology modeling predicts different loops in N. fischeri α-galactosidases, highlighting the larger tunnel structure in Gal27A to accommodate/bind branched galactomannan with high galactose contents. Phylogenetic analysis reveals the far relationship of Gal27A and Gal27B that they may evolve in different action modes, and their coexistence widens the substrate spectrum for nutrient utilization. This study illustrates the substrate profiles and synergistic mechanism of GH27 α-galactosidases of different structures.

Glutathionyl-hydroquinone reductases from poplar are plastidial proteins that deglutathionylate both reduced and oxidized glutathionylated quinones

Glutathionyl-hydroquinone reductases (GHRs) catalyze the deglutathionylation of quinones via a catalytic cysteine. The two GHR genes in the Populus trichocarpa genome, Pt-GHR1 and Pt-GHR2, are primarily expressed in reproductive organs. Both proteins are localized in plastids. More specifically, Pt-GHR2 localizes in nucleoids. At the structural level, Pt-GHR1 adopts a typical GHR fold, with a dimerization interface comparable to that of the bacterial and fungal GHR counterparts. Pt-GHR1 catalyzes the deglutathionylation of both reduced and oxidized glutathionylated quinones, but the enzyme is more catalytically efficient with the reduced forms.

Functional diversification of two UGT80 enzymes required for steryl glucoside synthesis in Arabidopsis.

Steryl glucosides (SG) are abundant steroid conjugates in plant membranes. Beyond structural roles in lipid bilayers, functions in sugar transport, storage, and/or signalling are predicted. UDP-glucose:sterol glucosyltransferase 80A2 (UGT80A2) and UGT80B1, which share similarity to fungal counterparts, are implicated in SG synthesis in Arabidopsis thaliana. A third related enzyme, which seems specific to the plant lineage, is encoded by UGT713B1/At5g24750. Genetic and biochemical approaches were employed to determine the role of each UGT gene in the production of specific SGs and acyl SGs (ASGs). Using direct infusion electrospray ionization tandem mass spectrometry (ESI-MS/MS), SG and acyl SG (ASG) contents of ugt80 and ugt713 mutants, and triple and double mutants were profiled in seeds. In vitro enzyme assays were performed to assay substrate preferences. Both UGT80A2 and UGT80B1, but not UGT713B1 were shown to be coordinately down-regulated during seed imbibition when SG levels decline, consistent with similar functions as UGT80 enzymes. UGT80A2 was found to be required for normal levels of major SGs in seeds, whereas UGT80B1 is involved in accumulation of minor SG and ASG compounds. Although the results demonstrate specific activities for UGT80A2 and UGT80B1, a role for UGT713B1 in SG synthesis was not supported. The data show that UGT80A2, the more highly conserved enzyme, is responsible for the bulk production of SGs in seeds, whereas UGT80B1 plays a critical accessory role. This study extends our knowledge of UGT80 enzymes and provides evidence for specialized functions for distinct classes of SG and ASG molecules in plants.

Purification and characterization of recombinant Cel7A from maize seed.

The corn grain biofactory was used to produce Cel7A, an exo-cellulase (cellobiohydrolase I) from Hypocrea jecorina. The enzymatic activity on small molecule substrates was equivalent to its fungal counterpart. The corn grain-derived enzyme is glycosylated and 6 kDa smaller than the native fungal protein, likely due to more sugars added in the glycosylation of the fungal enzyme. Our data suggest that corn seed-derived cellobiohydrolase (CBH) I performs as well as or better than its fungal counterpart in releasing sugars from complex substrates such as pre-treated corn stover or wood. This recombinant protein product can enter and expand current reagent enzyme markets as well as create new markets in textile or pulp processing. The purified protein is now available commercially.

Extracellular signals and receptor-like kinases regulating ROP GTPases in plants.

Rho-like GTPase from plants (ROPs) function as signaling switches that control a wide variety of cellular functions and behaviors including cell morphogenesis, cell division and cell differentiation. The Arabidopsis thaliana genome encodes 11 ROPs that form a distinct single subfamily contrarily to animal or fungal counterparts where multiple subfamilies of Rho GTPases exist. Since Rho proteins bind to their downstream effector proteins only in their GTP-bound “active” state, the activation of ROPs by upstream factor(s) is a critical step in the regulation of ROP signaling. Therefore, it is critical to examine the input signals that lead to the activation of ROPs. Recent findings showed that the plant hormone auxin is an important signal for the activation of ROPs during pavement cell morphogenesis as well as for other developmental processes. In contrast to auxin, another plant hormone, abscisic acid, negatively regulates ROP signaling. Calcium is another emerging signal in the regulation of ROP signaling. Several lines of evidence indicate that plasma membrane localized-receptor like kinases play a critical role in the transmission of the extracellular signals to intracellular ROP signaling pathways. This review focuses on how these signals impinge upon various direct regulators of ROPs to modulate various plant processes.

Molecular characterization of a highly-active thermophilic β-glucosidase from Neosartorya fischeri P1 and its application in the hydrolysis of soybean isoflavone glycosides.

Isoflavone occurs abundantly in leguminous seeds in the form of glycoside and aglycone. However, isoflavone glycoside has anti-nutritional effect and only the free type is beneficial to human health. In the present study we identified a β-glucosidase from thermophilic Neosartorya fischeri P1, termed NfBGL1, capable of efficiently converting isoflavone glycosides into free isoflavones. The gene, belonging to glycoside hydrolase family 3, was successfully overexpressed in Pichia pastoris at high cell density in a 3.7-l fermentor. Purified recombinant NfBGL1 had higher specific activity (2189±1.7 U/mg) and temperature optimum (80°C) than other fungal counterparts when using p-nitrophenyl β-d-glucopyranoside as the substrate. It retained stable at temperatures up to 70°C and over a broad pH range of 3.0−10.0. NfBGL1 had broad substrate specificity including glucosidase, cellobiase, xylanase and glucanase activities, and displayed preference for hydrolysis of β-1,2 glycosidic bond rather than β-1,3, β-1,4, β-1,6 bonds. The enzyme showed high bioconversion ability for major soybean isoflavone glycosides (daidin, gensitin and glycitin) into free forms. These properties make NfBGL1 potential for the wide use in the food, feed, pharmacy and biofuel industries.

Horizontal gene transfer and functional diversification of plant cell wall degrading polygalacturonases: Key events in the evolution of herbivory in beetles

Plant cell walls are the largest reservoir of organic carbon on earth. To breach and utilize this carbohydrate-rich protective barrier, microbes secrete plant cell wall degrading enzymes (PCWDEs) targeting pectin, cellulose and hemicelluloses. There is a growing body of evidence that genomes of some herbivorous insects also encode PCWDEs, raising questions about their evolutionary origins and functions. Among herbivorous beetles, pectin-degrading polygalacturonases (PGs) are found in the diverse superfamilies Chrysomeloidea (leaf beetles, long-horn beetles) and Curculionoidea (weevils). Here our aim was to test whether these arose from a common ancestor of beetles or via horizontal gene transfer (HGT), and whether PGs kept their ancestral function in degrading pectin or evolved novel functions. Transcriptome data derived from 10 beetle species were screened for PG-encoding sequences and used for phylogenetic comparisons with their bacterial, fungal and plant counterparts. These analyses revealed a large family of PG-encoding genes of Chrysomeloidea and Curculionoidea sharing a common ancestor, most similar to PG genes of ascomycete fungi. In addition, 50 PGs from beetle digestive systems were heterologously expressed and functionally characterized, showing a set of lineage-specific consecutively pectin-degrading enzymes, as well as conserved but enzymatically inactive PG proteins. The evidence indicates that a PG gene was horizontally transferred ∼200 million years ago from an ascomycete fungus to a common ancestor of Chrysomeloidea and Curculionoidea. This has been followed by independent duplications in these two lineages, as well as independent replacement in two sublineages of Chrysomeloidea by two other subsequent HGTs. This origin, leading to subsequent functional diversification of the PG gene family within its new hosts, was a key event promoting the evolution of herbivory in these beetles.

Impact of Matric Potential and Pore Size Distribution on Growth Dynamics of Filamentous and Non-Filamentous Soil Bacteria

The filamentous growth form is an important strategy for soil microbes to bridge air-filled pores in unsaturated soils. In particular, fungi perform better than bacteria in soils during drought, a property that has been ascribed to the hyphal growth form of fungi. However, it is unknown if, and to what extent, filamentous bacteria may also display similar advantages over non-filamentous bacteria in soils with low hydraulic connectivity. In addition to allowing for microbial interactions and competition across connected micro-sites, water films also facilitate the motility of non-filamentous bacteria. To examine these issues, we constructed and characterized a series of quartz sand microcosms differing in matric potential and pore size distribution and, consequently, in connection of micro-habitats via water films. Our sand microcosms were used to examine the individual and competitive responses of a filamentous bacterium (Streptomyces atratus) and a motile rod-shaped bacterium (Bacillus weihenstephanensis) to differences in pore sizes and matric potential. The Bacillus strain had an initial advantage in all sand microcosms, which could be attributed to its faster growth rate. At later stages of the incubation, Streptomyces became dominant in microcosms with low connectivity (coarse pores and dry conditions). These data, combined with information on bacterial motility (expansion potential) across a range of pore-size and moisture conditions, suggest that, like their much larger fungal counterparts, filamentous bacteria also use this growth form to facilitate growth and expansion under conditions of low hydraulic conductivity. The sand microcosm system developed and used in this study allowed for precise manipulation of hydraulic properties and pore size distribution, thereby providing a useful approach for future examinations of how these properties influence the composition, diversity and function of soil-borne microbial communities.

Loop Motions Important to Product Expulsion in the Thermobifida fusca Glycoside Hydrolase Family 6 Cellobiohydrolase from Structural and Computational Studies

Cellobiohydrolases (CBHs) are typically major components of natural enzyme cocktails for biomass degradation. Their active sites are enclosed in a tunnel, enabling processive hydrolysis of cellulose chains. Glycoside hydrolase Family 6 (GH6) CBHs act from nonreducing ends by an inverting mechanism and are present in many cellulolytic fungi and bacteria. The bacterial Thermobifida fusca Cel6B (TfuCel6B) exhibits a longer and more enclosed active site tunnel than its fungal counterparts. Here, we determine the structures of two TfuCel6B mutants co-crystallized with cellobiose, D274A (catalytic acid), and the double mutant D226A/S232A, which targets the putative catalytic base and a conserved serine that binds the nucleophilic water. The ligand binding and the structure of the active site are retained when compared with the wild type structure, supporting the hypothesis that these residues are directly involved in catalysis. One structure exhibits crystallographic waters that enable construction of a model of the α-anomer product after hydrolysis. Interestingly, the product sites of TfuCel6B are completely enclosed by an "exit loop" not present in fungal GH6 CBHs and by an extended "bottom loop". From the structures, we hypothesize that either of the loops enclosing the product subsites in the TfuCel6B active site tunnel must open substantially for product release. With simulation, we demonstrate that both loops can readily open to allow product release with equal probability in solution or when the enzyme is engaged on cellulose. Overall, this study reveals new structural details of GH6 CBHs likely important for functional differences among enzymes from this important family.

Tim50 in Trypanosoma brucei Possesses a Dual Specificity Phosphatase Activity and Is Critical for Mitochondrial Protein Import

In eukaryotes, proteins are imported into mitochondria via multiprotein translocases of the mitochondrial outer and inner membranes, TOM and TIM, respectively. Trypanosoma brucei, a hemoflagellated parasitic protozoan and the causative agent of African trypanosomiasis, imports about a thousand proteins into the mitochondrion; however, the mitochondrial protein import machinery in this organism is largely unidentified. Here, we characterized a homolog of Tim50 that is localized in the mitochondrial membrane in T. brucei. Similar to Tim50 proteins from fungi and mammals, Tim50 in T. brucei (TbTim50) possesses a mitochondrial targeting signal at its N terminus and a C-terminal domain phosphatase motif at its C terminus. Knockdown of TbTim50 reduced cell growth and inhibited import of proteins that contain N-terminal targeting signals. Co-immunoprecipitation analysis revealed that TbTim50 interacts with TbTim17. Unlike its fungal counterpart but similar to the human homolog of Tim50, recombinant TbTim50 possesses a dual specificity phosphatase activity with a greater affinity for protein tyrosine phosphate than for protein serine/threonine phosphate. Mutation of the aspartic acid residues to alanine in the C-terminal domain phosphatase motif (242)DXDX(V/T)(246) abolished activity for both type of substrates. TbTim50 knockdown increased and its overexpression decreased the level of voltage-dependent anion channel (VDAC). However, the VDAC level was unaltered when the phosphatase-inactive mutant of TbTim50 was overexpressed, suggesting that the phosphatase activity of TbTim50 plays a role in regulation of VDAC expression. In contrast, phosphatase activity of the TbTim50 is required neither for mitochondrial protein import nor for its interaction with TbTim17. Overall, our results show that TbTim50 plays additional roles in mitochondrial activities besides preprotein translocation.

Molecular Cloning and Expression of a Novel β-Glucosidase Gene from Phialophora sp. G5

A novel β-glucosidase gene, bgl1G5, was cloned from Phialophora sp. G5 and successfully expressed in Pichia pastoris. Sequence analysis indicated that the gene consists of a 1,431-bp open reading frame encoding a protein of 476 amino acids. The deduced amino acid sequence of bgl1G5 showed a high identity of 85% with a characterized β-glucosidase from Humicola grisea of glycoside hydrolase family 1. Compared with other fungal counterparts, Bgl1G5 showed similar optimal activity at pH 6.0 and 50 °C and was stable at pH 5.0-9.0. Moreover, Bgl1G5 exhibited good thermostability at 50 °C (6 h half-life) and higher specific activity (54.9 U mg⁻¹). The K (m) and V (max) values towards p-nitrophenyl β-D-glucopyranoside (pNPG) were 0.33 mM and 103.1 μmol min⁻¹ mg⁻¹, respectively. The substrate specificity assay showed that Bgl1G5 was highly active against pNPG, weak on p-nitrophenyl β-D-cellobioside (pNPC) and p-nitrophenyl-β-D-galactopyranoside (ONPG), and had no activity on cellobiose. This result indicated Bgl1G5 was a typical aryl β-glucosidase.

The Structure of a Bacterial Cellobiohydrolase: The Catalytic Core of the Thermobifida fusca Family GH6 Cellobiohydrolase Cel6B

Cellulases, glycoside hydrolases that catalyze the degradation of cellulose, are classified as either endoglucanases or cellobiohydrolases (CBHs) based on their architecture and mode of action on the cellulose. CBHs bind the cellulose chain in a more or less closed tunnel and cleave off cellobiose units processively from one end of the cellulosic polymer, while endoglucanases have their active sites in a more or less open cleft and show a higher tendency to cut bonds internally in the polymer. The CBH Cel6A (also called CBH2) from the ascomycete Hypocrea jecorina has a much shorter substrate-binding tunnel and seems less processive than the CBH Cel7A (CBH1), from the same fungus. Here, we present the X-ray crystal structure of the catalytic domain of the CBH Cel6B, also called E3, from the soil bacterium Thermobifida fusca, both in its apo form and co-crystallized with cellobiose. The enzyme structure reveals that the Cel6B enzyme has a much longer substrate-binding site than its fungal GH6 counterparts. The tunnel is comparable in length to that of GH7 CBHs. In the ligand structure with cellobiose, the tunnel exit is completely closed by a 13-residue loop not present in fungal GH6 enzymes. The loop needs to be displaced to allow cellobiose product release for a processive action by the enzyme. When ligand is absent, seven of these residues are not visible in the electron density and the tunnel exit is open.

Characterization of the Chitinolytic Machinery of Enterococcus faecalis V583 and High-Resolution Structure of Its Oxidative CBM33 Enzyme

Little information exists for the ability of enterococci to utilize chitin as a carbon source. We show that Enterococcus faecalis V583 can grow on chitin, and we describe two proteins, a family 18 chitinase (ef0361; EfChi18A) and a family 33 CBM (carbohydrate binding module) (ef0362; EfCBM33A) that catalyze chitin conversion in vitro. Various types of enzyme activity assays showed that EfChi18A has functional properties characteristic of an endochitinase. EfCBM33A belongs to a recently discovered family of enzymes that cleave glycosidic bonds via an oxidative mechanism and that act synergistically with classical hydrolytic enzymes (i.e., chitinases). The structure and function of this protein were probed in detail. An ultra-high-resolution crystal structure of EfCBM33A revealed details of a conserved binding surface that is optimized to interact with chitin and contains the catalytic center. Chromatography and mass spectrometry analyses of product formation showed that EfCBM33A cleaves chitin via the oxidative mechanism previously described for CBP21 from Serratia marcescens. Metal-depletion studies showed that EfCBM33A is a copper enzyme. In the presence of an external electron donor, EfCBM33A boosted the activity of EfChi18A, and combining the two enzymes led to rapid and complete conversion of β-chitin to chitobiose. This study provides insight into the structure and function of the CBM33 family of enzymes, which, together with their fungal counterpart called GH61, currently receive considerable attention in the biomass processing field.

A Novel Mechanism of P-type ATPase Autoinhibition Involving Both Termini of the Protein

The activity of many P-type ATPases is found to be regulated by interacting proteins or autoinhibitory elements located in N- or C-terminal extensions. An extended C terminus of fungal and plant P-type plasma membrane H(+)-ATPases has long been recognized to be part of a regulatory apparatus involving an autoinhibitory domain. Here we demonstrate that both the N and the C termini of the plant plasma membrane H(+)-ATPase are directly involved in controlling the pump activity state and that N-terminal displacements are coupled to secondary modifications taking place at the C-terminal end. This identifies the first group of P-type ATPases for which both ends of the polypeptide chain constitute regulatory domains, which together contribute to the autoinhibitory apparatus. This suggests an intricate mechanism of cis-regulation with both termini of the protein communicating to obtain the necessary control of the enzyme activity state.