The quaternary structure of the amidase from Geobacillus pallidus RAPc8 is revealed by its crystal packing

The amidase from Geobacillus pallidus RAPc8, a moderate thermophile, is a member of the nitrilase enzyme superfamily. It converts amides to the corresponding acids and ammonia and has application as an industrial catalyst. RAPc8 amidase has been cloned and functionally expressed in Escherichia coli and has been purified by heat treatment and a number of chromatographic steps. The enzyme was crystallized using the hanging-drop vapour-diffusion method. Crystals produced in the presence of 1.2 M sodium citrate, 400 mM NaCl, 100 mM sodium acetate pH 5.6 were selected for X-ray diffraction studies. A data set having acceptable statistics to 1.96 Å resolution was collected under cryoconditions using an in-house X-ray source. The space group was determined to be primitive cubic P4232, with unit-cell parameter a = 130.49 (±0.05) Å. The structure was solved by molecular replacement using the backbone of the hypothetical protein PH0642 from Pyrococcus horikoshii (PDB code 1j31 ) with all non-identical side chains substituted with alanine as a probe. There is one subunit per asymmetric unit. The subunits are packed as trimers of dimers with D3 point-group symmetry around the threefold axis in such a way that the dimer interface seen in the homologues is preserved

Sorghum grain mold: resistance stability in advanced B-lines

Grain mold resistance breeding in sorghum (Sorghum bicolor) at ICRISAT and in Indian national programs has focused on developing varieties, restorer lines, and hybrid seed parents utilizing resistance from germplasm lines of diverse geographical origin. During the past few years, ICRISAT has developed a large number of high-yielding, grain mold resistant B-llnes using pedigree breeding with single- and three-way crosses and selecting the progenies under high disease pressure In field screenings (Reddy et al. 2000). Resistance stability of some selected elite B-llnes was tested through a collaborative Sorghum Grain Mold Resistance Stability Nursery (SGMRSN) established In 2002. The results of trials conducted at diverse locations In India are presented

Sorghum grain mold: variability in fungal complex

The grain mold complex in sorghum {Sorghum bicolor) involves a number of pathogenic and saprophytic fungi that vary in their frequencies and severities under different environmental conditions (Bandyopadhyay et al. 2000). To provide genetic management for grain mold In sorghum, a clear understanding of the major pathogenic fungi and their variability under different environments is critical. Among the major pathogenic fungi, Fusarium monitiforme (F. verticiOoides) is known to produce fumonisins, a mycotoxin of concern for the use of molded sorghum grains as food and feed (Marasas 1996, Bhat et al. 1997). With the above objective we Initiated a collaborative Sorghum Grain Mold Variability Nursery (SGMVN) between ICRISAT and the All India Coordinated Sorghum Improvement Project (AICSIP) ot the Indian Council of Agricultural Research (ICAR). The nursery was coordinated by ICRISAT and conducted at four locations In India during the rainy season 2002. The results of the trials are presented

Variation in occurrence and severity of major sorghum grain mold pathogens in India

The variation in the occurrence and severity of pathogenic grain mould fungi were studied through a collaborative study in a Sorghum Grain Mould Variability Nursery (SGMVN), consisting of 12 sorghum genotypes, that was established at five locations (Akola and Parbhani, Maharashtra; Palem; Patancheru, Andhra Pradesh; and Surat, Gujarat) during the three rainy seasons of 2002-04. Grain mould infection severity by the major pathogens was recorded at physiological maturity and on threshed grain, and grain colonization was measured using the blotter method. Among the fungal species, Fusarium spp., Curvularia lunata [Cochliobolus lunatus], Alternaria alternata and Phoma sorghina, in receding order, were predominant across locations and genotypes. Analysis of variance indicated highly significant effects of location, year, genotype and their interactions on grain mould severity and grain colonization by the four fungi. Grain colonization was highest with Fusarium spp. at Parbhani (54%); with Curvularia lunata at Surat (45%); with A. alternata both at Parbhani (25%) and Patancheru (23%); and with P. sorghina at Patancheru (17%). Four of the sorghum genotypes (ICSV 96101, ICSV 95001, SPV 351/ICSV 1, and ICSV 91008) showed tolerance to mould (≤3.0 severity) and these could serve as sources of grain mould resistance

Evaluation of advanced sorghum breeding lines for grain mold resistance.

Grain mold, caused by a fungal complex, is a major production constraint of early-maturing high-yielding rainy season sorghum (Sorghum bicolor) hybrids. A total of 34 selected elite B-lines bred for grain mold resistance at ICRISAT were evaluated for their resistance stability through a collaborative Sorghum Grain Mold Resistance Stability Nursery under natural infection at five locations (Akola, Parbhani, Palem, Patancheru, and Surat) in India during three rainy seasons, 2002 to 2004. Grain mold severity scores were recorded at two stages, first at physiological maturity-panicle grain mold rating (PGMR) in the field nursery and second after harvest on threshed grain-threshed grain mold rating (TGMR), using a progressive 1-5 scale. Results indicated significant differences among the genotypes (G), locations (L), years (Y) and their interactions for both PGMR and TGMR scores. A strong positive correlation (r=0.89) between PGMR and TGMR indicated the adequacy of grain mold severity recording, preferably PGMR. Relatively, larger variance due to G than to G × L and G × Y interaction components justified breeding sorghum hybrid for grain mold resistance for wider adaptation in India

Structure of an aliphatic amidase from Geobacillus pallidus RAPc8

The amidase from Geobacillus pallidus RAPc8, a moderate thermophile, is a member of the nitrilase superfamily and catalyzes the conversion of amides to the corresponding carboxylic acids and ammonia. It shows both amide-hydrolysis and acyl-transfer activities and also exhibits stereoselectivity for some enantiomeric substrates, thus making it a potentially important industrial catalyst. The crystal structure of G. pallidus RAPc8 amidase at a resolution of 1.9 Å was solved by molecular replacement from a crystal belonging to the primitive cubic space group P4232. G. pallidus RAPc8 amidase is homohexameric in solution and its monomers have the typical nitrilase-superfamily [alpha]-[beta]-[beta]-[alpha] fold. Association in the hexamer preserves the eight-layered [alpha]-[beta]-[beta]-[alpha]:[alpha]-[beta]-[beta]-[alpha] structure across an interface which is conserved in the known members of the superfamily. The extended carboxy-terminal tail contributes to this conserved interface by interlocking the monomers. Analysis of the small active site of the G. pallidus RAPc8 amidase suggests that access of a water molecule to the catalytic triad (Cys, Glu, Lys) side chains would be impeded by the formation of the acyl intermediate. It is proposed that another active-site residue, Glu142, the position of which is conserved in the homologues, acts as a general base to catalyse the hydrolysis of this intermediate. The small size of the substrate-binding pocket also explains the specificity of this enzyme for short aliphatic amides and its asymmetry explains its enantioselectivity

A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity

An amidase (EC 3.5.1.4) in branch 2 of the nitrilase superfamily, from the thermophilic strain Geobacillus pallidus RAPc8, was produced at high expression levels (20 U/mg) in small-scale fermentations of Escherichia coli. The enzyme was purified to 90% homogeneity with specific activity of 1,800 U/mg in just two steps, namely, heat-treatment and gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electron microscopic (EM) analysis of the homogenous enzyme showed the native enzyme to be a homohexamer of 38 kDa subunits. Analysis of the biochemical properties of the amidase showed that the optimal temperature and pH for activity were 50 and 7.0°C, respectively. The amidase exhibited high thermal stability at 50 and 60°C, with half-lives greater than 5 h at both temperatures. At 70 and 80°C, the half-life values were 43 and 10 min, respectively. The amidase catalyzed the hydrolysis of low molecular weight aliphatic amides, with d-selectivity towards lactamide. Inhibition studies showed activation/inhibition data consistent with the presence of a catalytically active thiol group. Acyl transfer reactions were demonstrated with acetamide, propionamide, isobutyramide, and acrylamide as substrates and hydroxylamine as the acyl acceptor; the highest reaction rate being with isobutyramide. Immobilization by entrapment in polyacrylamide gels, covalent binding on Eupergit C beads at 4°C and on Amberlite-XAD57 resulted in low protein binding and low activity, but immobilization on Eupergit C beads at 25°C with cross-linking resulted in high protein binding yield and high immobilized specific activity (80% of non-immobilized activity). Characterization of Eupergit C-immobilized preparations showed that the optimum reaction temperature was unchanged, the pH range was somewhat broadened, and stability was enhanced giving half-lives of 52 min at 70°C and 30 min at 80°C. The amidase has potential for application under high temperature conditions as a biocatalyst for d-selective amide hydrolysis producing enantiomerically pure carboxylic acids and for production of novel amides by acyl transfer