Sugarcane (Saccharum spp.) is a clonally propagated outcrossing polyploid crop of great importance in tropical agriculture. Up to now, all sugarcane genetic maps had been developed using either full-sib progenies derived from interspecific crosses or from selfing, both approaches not directly adopted in conventional breeding. We have developed a single integrated genetic map using a population derived from a cross between two pre-commercial cultivars ('SP80-180' x 'SP80-4966') using a novel approach based on the simultaneous maximum-likelihood estimation of linkage and linkage phases method specially designed for outcrossing species. From a total of 1,118 single-dose markers (RFLP, SSR and AFLP) identified, 39% derived from a testcross configuration between the parents segregating in a 1:1 fashion, while 61% segregated 3:1, representing heterozygous markers in both parents with the same genotypes. The markers segregating 3:1 were used to establish linkage between the testcross markers. The final map comprised of 357 linked markers, including 57 RFLPs, 64 SSRs and 236 AFLPs that were assigned to 131 co-segregation groups, considering a LOD score of 5, and a recombination fraction of 37.5 cM with map distances estimated by Kosambi function. The co-segregation groups represented a total map length of 2,602.4 cM, with a marker density of 7.3 cM. When the same data were analyzed using JoinMap software, only 217 linked markers were assigned to 98 co-segregation groups, spanning 1,340 cM, with a marker density of 6.2 cM. The maximum-likelihood approach reduced the number of unlinked markers to 761 (68.0%), compared to 901 (80.5%) using JoinMap. All the co-segregation groups obtained using JoinMap were present in the map constructed based on the maximum-likelihood method. Differences on the marker order within the co-segregation groups were observed between the two maps. Based on RFLP and SSR markers, 42 of the 131 co-segregation groups were assembled into 12 putative homology groups. Overall, the simultaneous maximum-likelihood estimation of linkage and linkage phases was more efficient than the method used by JoinMap to generate an integrated genetic map of sugarcane.

Cellulases participate in a number of biological events, such as plant cell wall remodelling, nematode parasitism and microbial carbon uptake. Their ability to depolymerize crystalline cellulose is of great biotechnological interest for environmentally compatible production of fuels from lignocellulosic biomass. However, industrial use of cellulases is somewhat limited by both their low catalytic efficiency and stability. In the present study, we conducted a detailed functional and structural characterization of the thermostable BsCel5A (Bacillus subtilis cellulase 5A), which consists of a GH5 (glycoside hydrolase 5) catalytic domain fused to a CBM3 (family 3 carbohydrate-binding module). NMR structural analysis revealed that the Bacillus CBM3 represents a new subfamily, which lacks the classical calcium-binding motif, and variations in NMR frequencies in the presence of cellopentaose showed the importance of polar residues in the carbohydrate interaction. Together with the catalytic domain, the CBM3 forms a large planar surface for cellulose recognition, which conducts the substrate in a proper conformation to the active site and increases enzymatic efficiency. Notably, the manganese ion was demonstrated to have a hyper-stabilizing effect on BsCel5A, and by using deletion constructs and X-ray crystallography we determined that this effect maps to a negatively charged motif located at the opposite face of the catalytic site.

Background: Arabinanases are key enzymes involved in hemicellulose degradation. Results: Crystallographic, mutational, and biochemical assays of three arabinanases reveal the molecular mechanisms governing their catalysis and activation. Conclusion: Accessory domain and metal ion are essential for catalysis. Structural adaptations in the catalytic interface confer unique action modes to ruminal arabinanases. Significance: This work provides new molecular strategies for arabinan hydrolysis.

The breakdown of β-1,4-mannoside linkages in a variety of mannan-containing polysaccharides is of great importance in industrial processes such as kraft pulp delignification, food processing and production of second-generation biofuels, which puts a premium on studies regarding the prospection and engineering of β-mannanases. In this work, a two-domain β-mannanase from Thermotoga petrophila that encompasses a GH5 catalytic domain with a C-terminal CBM27 accessory domain, was functionally and structurally characterized. Kinetic and thermal denaturation experiments showed that the CBM27 domain provided thermo-protection to the catalytic domain, while no contribution on enzymatic activity was observed. The structure of the catalytic domain determined by SIRAS revealed a canonical (α/β)(8)-barrel scaffold surrounded by loops and short helices that form the catalytic interface. Several structurally related ligand molecules interacting with TpMan were solved at high-resolution and resulted in a wide-range representation of the subsites forming the active-site cleft with residues W134, E198, R200, E235, H283 and W284 directly involved in glucose binding.

Disulfide oxidoreductase DsbA catalyzes disulfide bond formation in proteins secreted to the periplasm and has been related to the folding process of virulence factors in many organisms. It is among the most oxidizing of the thioredoxin-like proteins, and DsbA redox power is understood in terms of the electrostatic interactions involving the active site motif CPHC. The plant pathogen Xylella fastidiosa has two chromosomal genes encoding two oxidoreductases belonging to the DsbA family, and in one of them, the canonical motif CPHC is replaced by CPAC. Biochemical assays showed that both X. fastidiosa homologues have similar redox properties and the determination of the crystal structure of XfDsbA revealed substitutions in the active site of X. fastidiosa enzymes, which are proposed to compensate for the lack of the conserved histidine in XfDsbA2. In addition, electron density maps showed a ligand bound to the XfDsbA active site, allowing the characterization of the enzyme interaction with an 8-mer peptide. Finally, surface analysis of XfDsbA and XfDsbA2 suggests that X. fastidiosa enzymes may have different substrate specificities.

Endo-xylanases play a key role in the depolymerization of xylan and recently, they have attracted much attention owing to their potential applications on biofuels and paper industries. In this work, we have investigated the molecular basis for the action mode of xylanases 10B at high temperatures using biochemical, biophysical and crystallographic methods. The crystal structure of xylanase 10B from hyperthermophilic bacterium Thermotoga petrophila RKU-1 (TpXyl10B) has been solved in the native state and in complex with xylobiose. The complex crystal structure showed a classical binding mode shared among other xylanases, which encompasses the -1 and -2 subsites. Interestingly, TpXyl10B displayed a temperature-dependent action mode producing xylobiose and xylotriose at 20°C, and exclusively xylobiose at 90°C as assessed by capillary zone electrophoresis. Moreover, circular dichroism spectroscopy suggested a coupling effect of temperature-induced structural changes with this particular enzymatic behavior. Molecular dynamics simulations supported the CD analysis suggesting that an open conformational state adopted by the catalytic loop (Trp297-Lys326) provokes significant modifications in the product release area (+1,+2 and +3 subsites), which drives the enzymatic activity to the specific release of xylobiose at high temperatures.

The hexameric purine nucleoside phosphorylase from Bacillus subtilis (BsPNP233) displays great potential to produce nucleoside analogues in industry and can be exploited in the development of new anti-tumor gene therapies. In order to provide structural basis for enzyme and substrates rational optimization, aiming at those applications, the present work shows a thorough and detailed structural description of the binding mode of substrates and nucleoside analogues to the active site of the hexameric BsPNP233. Here we report the crystal structure of BsPNP233 in the apo form and in complex with 11 ligands, including clinically relevant compounds. The crystal structure of six ligands (adenine, 2′deoxyguanosine, aciclovir, ganciclovir, 8-bromoguanosine, 6-chloroguanosine) in complex with a hexameric PNP are presented for the first time. Our data showed that free bases adopt alternative conformations in the BsPNP233 active site and indicated that binding of the co-substrate (2′deoxy)ribose 1-phosphate might contribute for stabilizing the bases in a favorable orientation for catalysis. The BsPNP233-adenosine complex revealed that a hydrogen bond between the 5′ hydroxyl group of adenosine and Arg43* side chain contributes for the ribosyl radical to adopt an unusual C3’-endo conformation. The structures with 6-chloroguanosine and 8-bromoguanosine pointed out that the Cl6 and Br8 substrate modifications seem to be detrimental for catalysis and can be explored in the design of inhibitors for hexameric PNPs from pathogens. Our data also corroborated the competitive inhibition mechanism of hexameric PNPs by tubercidin and suggested that the acyclic nucleoside ganciclovir is a better inhibitor for hexameric PNPs than aciclovir. Furthermore, comparative structural analyses indicated that the replacement of Ser90 by a threonine in the B. cereus hexameric adenosine phosphorylase (Thr91) is responsible for the lack of negative cooperativity of phosphate binding in this enzyme.

Cyanide is a toxic compound that is converted to the non-toxic thiocyanate by a rhodanese enzyme. Rhodaneses belong to the family of transferases (sulfurtransferases), which are largely studied. The sulfur donor defines the subfamily of these enzymes as thiosulfate:cyanide sulfurtransferases or rhodaneses (TSTs) or 3-mercaptopyruvate sulfurtransfeases (MSTs). In Mycobacterium tuberculosis, the causative agent of tuberculosis, the gene Rv0815c encodes the protein CysA2, a putative uncharacterized thiosulfate:cyanide sulfurtransferase that belongs to the essential sulfur assimilation pathway in the bacillus and is secreted during infection. In this work, we characterized the functional and structural properties of CysA2 and its kinetic parameters. The recombinant CysA2 is a α/β protein with two rhodanese-like domains that maintains the functional motifs and a catalytic cysteine. Sulfurtransferase activity was determined using thiosulfate and 3-mercaptopyruvate as sulfur donors. The assays showed Km values of 2.89 mM and 7.02 mM for thiosulfate and 3-mercaptopyruvate, respectively, indicating the protein has dual activity as TST and MST. Immunological assays revealed that CysA2 interacted with pulmonary cells, and it was capable to activate macrophages and dendritic cells, indicating the stimulation of the immune response, which is important for its use as an antigen for vaccine development and immunodiagnostic.