Wimal Ubhayasekera

The first crystal structures of a family 19 class IV chitinase: the enzyme from Norway spruce

Chitinases help plants defend themselves against fungal attack, and play roles in other processes, including development. The catalytic modules of most plant chitinases belong to glycoside hydrolase family 19. We report here x-ray structures of such a module from a Norway spruce enzyme, the first for any family 19 class IV chitinase. The bi-lobed structure has a wide cleft lined by conserved residues; the most interesting for catalysis are Glu113, the proton donor, and Glu122, believed to be a general base that activate a catalytic water molecule. Comparisons to class I and II enzymes show that loop deletions in the class IV proteins make the catalytic cleft shorter and wider; from modeling studies, it is predicted that only three N-acetylglucosamine-binding subsites exist in class IV. Further, the structural comparisons suggest that the family 19 enzymes become more closed on substrate binding. Attempts to solve the structure of the complete protein including the associated chitin-binding module failed, however, modeling studies based on close relatives indicate that the binding module recognizes at most three N-acetylglucosamine units. The combined results suggest that the class IV enzymes are optimized for shorter substrates than the class I and II enzymes, or alternatively, that they are better suited for action on substrates where only small regions of chitin chain are accessible. Intact spruce chitinase is shown to possess antifungal activity, which requires the binding module; removing this module had no effect on measured chitinase activity.

A missense mutation in ftsZ differentially affects vegetative and developmentally controlled cell division in Streptomyces coelicolor A3(2)

Streptomyces coelicolor A3(2) undergoes at least two kinds of cell division: vegetative septation leading to cross‐walls in the substrate mycelium; and developmentally regulated sporulation septation in aerial hyphae. By isolation and characterization of a non‐sporulating ftsZ mutant, we demonstrate a difference between the two types of septation. The ftsZ17 (Spo) allele gave rise to a classical white phenotype. The mutant grew as well as the parent on plates, and formed apparently normal hyphal cross‐walls, although with a small reduction in frequency. In contrast, sporulation septation was almost completely abolished, resulting in a phenotype reminiscent of whiH and ftsZΔ2p mutants. The ftsZ17 (Spo) allele was partially dominant and had no detectable effect on the cellular FtsZ content. As judged from both immunofluorescence microscopy of FtsZ and translational fusion of ftsZ to egfp , the mutation prevented correct temporal and spatial assembly of Z rings in sporulating hyphae. Homology modelling of S. coelicolor FtsZ indicated that the mutation, an A249T change in the C‐terminal domain, would be expected to alter the protein on the lateral face of FtsZ protofilaments. The results suggest that cytokinesis may be developmentally controlled at the level of Z‐ring assembly during sporulation of S. coelicolor A3(2).

Structures of Phanerochaete chrysosporium Cel7D in complex with product and inhibitors

The cellobiohydrolase Pc_Cel7D is the major cellulase produced by the white‐rot fungus Phanerochaete chrysosporium, constituting ≈10% of the total secreted protein in liquid culture on cellulose. The enzyme is classified into family 7 of the glycoside hydrolases and, like other family members, catalyses cellulose hydrolysis with net retention of the anomeric carbon configuration. Previous work described the apo structure of the enzyme. Here we investigate the binding of the product, cellobiose, and several inhibitors, i.e. lactose, cellobioimidazole, Tris/HCl, calcium and a thio‐linked substrate analogue, methyl 4‐S‐β‐cellobiosyl‐4‐thio‐β‐cellobioside (GG‐S‐GG). The three disaccharides bind in the glucosyl‐binding subsites +1 and +2, close to the exit of the cellulose‐binding tunnel/cleft. Pc_Cel7D binds to lactose more strongly than cellobiose, while the opposite is true for the homologous Trichoderma reesei cellobiohydrolase Tr_Cel7A. Although both sugars bind Pc_Cel7D in a similar fashion, the different preferences can be explained by varying interactions with nearby loops. Cellobioimidazole is bound at a slightly different position, displaced ≈2 Å toward the catalytic centre. Thus the Pc_Cel7D complexes provide evidence for two binding modes of the reducing‐end cellobiosyl moiety; this conclusion is confirmed by comparison with other available structures. The combined results suggest that hydrolysis of the glycosyl‐enzyme intermediate may not require the prior release of the cellobiose product from the enzyme. Further, the structure obtained in the presence of both GG‐S‐GG and cellobiose revealed electron density for Tris at the catalytic centre. Inhibition experiments confirm that both Tris and calcium are effective inhibitors at the conditions used for crystallization.

Crystal structures of a family 19 chitinase from Brassica juncea show flexibility of binding cleft loops

Brassica juncea chitinase is an endo‐acting, pathogenesis‐related protein that is classified into glycoside hydrolase family 19, with highest homology (50–60%) in its catalytic domain to class I plant chitinases. Here we report X‐ray structures of the chitinase catalytic domain from wild‐type (apo, as well as with chloride ions bound) and a Glu234Ala mutant enzyme, solved by molecular replacement and refined at 1.53, 1.8 and 1.7 Å resolution, respectively. Confirming our earlier mutagenesis studies, the active‐site residues are identified as Glu212 and Glu234. Glu212 is believed to be the catalytic acid in the reaction, whereas Glu234 is thought to have a dual role, both activating a water molecule in its attack on the anomeric carbon, and stabilizing the charged intermediate. The molecules in the various structures differ significantly in the conformation of a number of loops that border the active‐site cleft. The differences suggest an opening and closing of the enzyme during the catalytic cycle. Chitin is expected to dock first near Glu212, which will protonate it. Conformational changes then bring Glu234 closer, allowing it to assist in the following steps. These observations provide important insights into catalysis in family 19 chitinases.

Functional analyses of the chitin-binding domains and the catalytic domain of Brassica juncea chitinase BjCHI1

We previously isolated a Brassica juncea cDNA encoding BjCHI1, a novel chitinase with two chitin-binding domains. Synthesis of its mRNA is induced by wounding, methyl jasmonate treatment, Aspergillus niger infection and caterpillar Pieris rapae feeding, suggesting that the protein has a role in defense. In that it possesses two chitin-binding domains, BjCHI1 resembles the precursor of Urtica dioica agglutinin but unlike that protein, BjCHI1 retains its chitinase catalytic domain after post-translational processing. To explore the properties of multi-domain BjCHI1, we have expressed recombinant BjCHI1 and two derivatives, which lack one (BjCHI2) or both (BjCHI3) chitin-binding domains, as secreted proteins in Pichia pastoris. Recombinant BjCHI1 and BjCHI2, showed apparent molecular masses on SDS-PAGE larger than calculated, and could be deglycosylated using α-mannosidase. Recombinant BjCHI3, without the proline/threonine-rich linker region containing predicted O-glycosylation sites, did not appear to be processed by α-mannosidase. BjCHI1’s ability to agglutinate rabbit erythrocytes is unique among known chitinases. Both chitin-binding domains are essential for agglutination; this property is absent in recombinant BjCHI2 and BjCHI3. To identify potential catalytic residues, we generated site-directed mutations in recombinant BjCHI3. Mutation E212A showed the largest effect, exhibiting 0 of wild-type specific activity. H211N and R361A resulted in considerable (>91) activity loss, implying these charged residues are also important in catalysis. E234A showed 36 retention of activity and substitution Y269D, 50. The least affected mutants were E349A and D360A, with 73 and 68 retention, respectively. Like Y269, E349 and D360 are possibly involved in substrate binding rather than catalysis.

Cel6A, a major exoglucanase from the cellulosome of the anaerobic fungi Piromyces sp. E2 and Piromyces equi.

Anaerobic fungi possess high cellulolytic activities, which are organised in high molecular mass (HMM) complexes. Besides catalytic modules, the cellulolytic enzyme components of these complexes contain non-catalytic modules, known as dockerins, that play a key role in complex assembly. Screening of a genomic and a cDNA library of two Piromyces species resulted in the isolation of two clones containing inserts of 5.5 kb (Piromyces sp. E2) and 1.5 kb (Piromyces equi). Both clones contained the complete coding region of a glycoside hydrolase (GH) from family 6, consisting of a 20 amino acid signal peptide, a 76 (sp. E2)/81 (P. equi) amino acid stretch comprising two fungal non-catalytic docking domains (NCDDs), a 24 (sp. E2)/16 (P. equi) amino acid linker, and a 369 amino acid catalytic module. Homology modelling of the catalytic module strongly suggests that the Piromyces enzymes will be processive cellobiohydrolases. The catalytic residues and all nearby residues are conserved. The reaction is thus expected to proceed via a classical single-displacement (inverting) mechanism that is characteristic of this family of GHs. The enzyme, defined as Cel6A, encoded by the full-length Piromyces E2 sequence was expressed in Escherichia coli. The recombinant protein expressed had a molecular mass of 55 kDa and showed activity against Avicel, supporting the observed relationship of the sequence to those of known cellobiohydrolases. Affinity-purified cellulosomes of Piromyces sp. E2 were analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis. A major band was detected with the molecular weight of Cel6A. A tryptic fingerprint of this protein confirmed its identity.

An intron-containing glycoside hydrolase family 9 cellulase gene encodes the dominant 90 kDa component of the cellulosome of the anaerobic fungus Piromyces sp. strain E2.

The cellulosome produced by Piromyces sp. strain E2 during growth on filter paper was purified by using an optimized cellulose-affinity method consisting of steps of EDTA washing of the cellulose-bound protein followed by elution with water. Three dominant proteins were identified in the cellulosome preparation, with molecular masses of 55, 80 and 90 kDa. Treatment of cellulose-bound cellulosome with a number of denaturing agents was also tested. Incubation with 0.5% (w/v) SDS or 8 M urea released most cellulosomal proteins, while leaving the greater fraction of the 80, 90 and 170 kDa components. To investigate the major 90 kDa cellulosome protein further, the corresponding gene, cel9A, was isolated, using immunoscreening and N-terminal sequencing. Inspection of the cel9A genomic organization revealed the presence of four introns, allowing the construction of a consensus for introns in anaerobic fungi. The 2800 bp cDNA clone contained an open reading frame of 2334 bp encoding a 757-residue extracellular protein. Cel9A includes a 445-residue glycoside hydrolase family 9 catalytic domain, and so is the first fungal representative of this large family. Both modelling of the catalytic domain as well as the activity measured with low level expression in Escherichia coli indicated that Cel9A is an endoglucanase. The catalytic domain is succeeded by a putative beta-sheet module of 160 amino acids with unknown function, followed by a threonine-rich linker and three fungal docking domains. Homology modelling of the Cel9A dockerins suggested that the cysteine residues present are all involved in disulphide bridges. The results presented here are used to discuss evolution of glycoside hydrolase family 9 enzymes.

Conformational changes and ligand recognition of Escherichia coli D-xylose binding protein revealed

ATP binding cassette transport systems account for most import of necessary nutrients in bacteria. The periplasmic binding component (or an equivalent membrane-anchored protein) is critical to recognizing cognate ligand and directing it to the appropriate membrane permease. Here we report the X-ray structures of D-xylose binding protein from Escherichia coli in ligand-free open form, ligand-bound open form, and ligand-bound closed form at 2.15 Å, 2.2 Å, and 2.2 Å resolutions, respectively. The ligand-bound open form is the first such structure to be reported at high resolution; the combination of the three different forms from the same protein furthermore gives unprecedented details concerning the conformational changes involved in binding protein function. As is typical of the structural family, the protein has two similar globular domains, which are connected by a three-stranded hinge region. The open liganded structure shows that xylose binds first to the C-terminal domain, with only very small conformational changes resulting. After a 34° closing motion, additional interactions are formed with the N-terminal domain; changes in this domain are larger and serve to make the structure more ordered near the ligand. An analysis of the interactions suggests why xylose is the preferred ligand. Furthermore, a comparison with the most closely related proteins in the structural family shows that the conformational changes are distinct in each type of binding protein, which may have implications for how the individual proteins act in concert with their respective membrane permeases.

Evolutionary analysis of hydrophobin gene family in two wood-degrading basidiomycetes, Phlebia brevispora and Heterobasidion annosum s.l.

BackgroundHydrophobins are small secreted cysteine-rich proteins that play diverse roles during different phases of fungal life cycle. In basidiomycetes, hydrophobin-encoding genes often form large multigene families with up to 40 members. The evolutionary forces driving hydrophobin gene expansion and diversification in basidiomycetes are poorly understood. The functional roles of individual genes within such gene families also remain unclear. The relationship between the hydrophobin gene number, the genome size and the lifestyle of respective fungal species has not yet been thoroughly investigated. Here, we present results of our survey of hydrophobin gene families in two species of wood-degrading basidiomycetes, Phlebia brevispora and Heterobasidion annosum s.l. We have also investigated the regulatory pattern of hydrophobin-encoding genes from H. annosum s.s. during saprotrophic growth on pine wood as well as on culture filtrate from Phlebiopsis gigantea using micro-arrays. These data are supplemented by results of the protein structure modeling for a representative set of hydrophobins.ResultsWe have identified hydrophobin genes from the genomes of two wood-degrading species of basidiomycetes, Heterobasidion irregulare, representing one of the microspecies within the aggregate H. annosum s.l., and Phlebia brevispora. Although a high number of hydrophobin-encoding genes were observed in H. irregulare (16 copies), a remarkable expansion of these genes was recorded in P. brevispora (26 copies). A significant expansion of hydrophobin-encoding genes in other analyzed basidiomycetes was also documented (1–40 copies), whereas contraction through gene loss was observed among the analyzed ascomycetes (1–11 copies). Our phylogenetic analysis confirmed the important role of gene duplication events in the evolution of hydrophobins in basidiomycetes. Increased number of hydrophobin-encoding genes appears to have been linked to the species’ ecological strategy, with the non-pathogenic fungi having increased numbers of hydrophobins compared with their pathogenic counterparts. However, there was no significant relationship between the number of hydrophobin-encoding genes and genome size. Furthermore, our results revealed significant differences in the expression levels of the 16 H. annosum s.s. hydrophobin-encoding genes which suggest possible differences in their regulatory patterns.ConclusionsA considerable expansion of the hydrophobin-encoding genes in basidiomycetes has been observed. The distribution and number of hydrophobin-encoding genes in the analyzed species may be connected to their ecological preferences. Results of our analysis also have shown that H. annosum s.l. hydrophobin-encoding genes may be under positive selection. Our gene expression analysis revealed differential expression of H. annosum s.s. hydrophobin genes under different growth conditions, indicating their possible functional diversification.

Molecular Cloning of a Genomic DNA Encoding Yam Class IV Chitinase

Genomic DNA for a class IV chitinase was cloned from yam (Dioscorea opposita Thunb) leaves and sequenced. The deduced amino acid sequence shows 50 to 59% identity to class IV chitinases from other plants. The yam chitinase, however, has an additional sequence of 8 amino acids (a C-terminal extension) following the cysteine that was reported as the last amino acid for other class IV chitinases; this extension is perhaps involved in subcellular localization. A homology model based on the structure of a class II chitinase from barley was used as an aid to interpreting the available data. The analysis suggests that the class IV enzyme recognizes an even shorter segment of the substrate than class I or II enzymes. This observation might help to explain why class IV enzymes are better suited to attack against pathogen cell walls.