Warriors at the gate that never sleep: non-host resistance in plants

The native resistance of most plant species against a wide variety of pathogens is known as non-host resistance (NHR), which confers durable protection to plant species. Only a few pathogens or parasites can successfully cause diseases. NHR is polygenic and appears to be linked with basal plant resistance, a form of elicited protection. Sensing of pathogens by plants is brought about through the recognition of invariant pathogen-associated molecular patterns (PAMPs) that trigger downstream defense signaling pathways. Race-specific resistance, (R)-gene mediated resistance, has been extensively studied and reviewed, while our knowledge of NHR has advanced only recently due to the improved access to excellent model systems. The continuum of the cell wall (CW) and the CW-plasma membrane (PM)-cytoskeleton plays a crucial role in perceiving external cues and activating defense signaling cascades during NHR. Based on the type of hypersensitive reaction (HR) triggered, NHR was classified into two types, namely type-I and type-II. Genetic analysis of Arabidopsis mutants has revealed important roles for a number of specific molecules in NHR, including the role of SNARE-complex mediated exocytosis, lipid rafts and vesicle trafficking. As might be expected, R-gene mediated resistance is found to overlap with NHR, but the extent to which the genes/pathways are common between these two forms of disease resistance is unknown. The present review focuses on the various components involved in the known mechanisms of NHR in plants with special reference to the role of CW-PM components.     

A multi-faceted analysis of RutD reveals a novel family of α/β hydrolases

The rut pathway of pyrimidine catabolism is a novel pathway that allows pyrimidine bases to serve as the sole nitrogen source in suboptimal temperatures. The rut operon in E. coli evaded detection until 2006, yet consists of seven proteins named RutA, RutB, etc. through RutG. The operon is comprised of a pyrimidine transporter and six enzymes that cleave and further process the uracil ring. Herein, we report the structure of RutD, a member of the α/β hydrolase superfamily, which is proposed to enhance the rate of hydrolysis of aminoacrylate, a toxic side product of uracil degradation, to malonic semialdehyde. Although this reaction will occur spontaneously in water, the toxicity of aminoacrylate necessitates catalysis by RutD for efficient growth with uracil as a nitrogen source. RutD has a novel and conserved arrangement of residues corresponding to the α/β hydrolase active site, where the nucleophile's spatial position occupied by Ser, Cys, or Asp of the canonical catalytic triad is replaced by histidine. We have used a combination of crystallographic structure determination, modeling and bioinformatics, to propose a novel mechanism for this enzyme. This approach also revealed that RutD represents a previously undescribed family within the α/β hydrolases. We compare and contrast RutD with PcaD, which is the closest structural homolog to RutD. PcaD is a 3‐oxoadipate‐enol‐lactonase with a classic arrangement of residues in the active site. We have modeled a substrate in the PcaD active site and proposed a reaction mechanism. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Interspecies differences in the enantioselectivity of epoxide hydrolases in Cryptococcus laurentii (Kufferath) C.E. Skinner and Cryptococcus podzolicus (Bab'jeva & Reshetova) Golubev

Isolates representing Cryptococcus laurentii and Cryptococcus podzolicus, originating from soil of a heathland indigenous to South Africa, were screened for the presence of enantioselective epoxide hydrolases for 2,2-disubstituted epoxides. Epoxide hydrolase activity for the 2,2-disubstituted epoxide (+/-)-2-methyl-2-pentyl oxirane was found to be abundantly present in all isolates. The stereochemistry of the products formed by the epoxide hydrolase enzymes from isolates belonging to the two species (11 isolates representing C. laurentii and 23 isolates representing C. podzolicus) was investigated. The enantiopreferences of the epoxide hydrolases for 2,2-disubstituted epoxides of these two species were found to be opposite. All strains of C. laurentii preferentially hydrolysed the (S)-epoxides while all C. podzolicus isolates preferentially hydrolysed the (R)-epoxides of (+/-)-2,2-disubstituted epoxides. These findings indicate that the stereochemistry of the products formed from 2,2-disubstituted epoxides by the epoxide hydrolase enzymes of these yeasts should be evaluated as additional taxonomic criterion within the genus Cryptococcus. Also, the selectivity of some epoxide hydrolases originating from isolates of C. podzolicus was high enough to be considered for application in biotransformations for the synthesis of enantiopure epoxides and vicinal diols.     

DOM-fold: a structure with crossing loops found in DmpA, ornithine acetyltransferase, and molybdenum cofactor-binding domain

Understanding relationships between sequence, structure, and evolution is important for functional characterization of proteins. Here, we define a novel DOM-fold as a consensus structure of the domains in DmpA (L-aminopeptidase D-Ala-esterase/amidase), OAT (ornithine acetyltransferase), and MocoBD (molybdenum cofactor-binding domain), and discuss possible evolutionary scenarios of its origin. As shown by a comprehensive structure similarity search, DOM-fold distinguished by a two-layered beta/alpha architecture of a particular topology with unusual crossing loops is unique to those three protein families. DmpA and OAT are evolutionarily related as indicated by their sequence, structural, and functional similarities. Structural similarity between the DmpA/OAT superfamily and the MocoBD domains has not been reported before. Contrary to previous reports, we conclude that functional similarities between DmpA/OAT proteins and N-terminal nucleophile (Ntn) hydrolases are convergent and are unlikely to be inherited from a common ancestor.     

The autolytic phenotype of the Bacillus cereus group

AIM: To determine the autolytic phenotype of five species in the Bacillus cereus group. METHODS AND RESULTS: The autolytic rate of 96 strains belonging to five species in the B. cereus group was examined under starvation conditions at pH 6, 6.5 and 8.5 in different buffers. The autolytic rate was strain-dependent with a wide variability at pH 6, but higher and more uniform at pH 6.5. At pH 8.5, and respect to the extent of autolysis at pH 6.5, it was relatively low for most of the strains with the lowest values between 13 and 52% in Bacillus mycoides and Bacillus pseudomycoides. Peptidoglycan hydrolase patterns evaluated by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis using cells of Bacillus thuringiensis ssp. tolworthi HD125 as an indicator, revealed complex profiles with lytic bands of about 90, 63, 46, 41, 38, 32, 28 and 25 kDa in B. cereus, B. thuringiensis and Bacillus weihenstephanensis. Bacillus mycoides and B. pseudomycoides had simpler profiles with lytic bands of 63, 46 and 38 kDa. Changes in the autolytic pattern were observed for cells harvested at the stationary phase of growth (72 h) showing an increase in the intensity of the 25 kDa band in the case of B. cereus, B. thuringiensis and B. weihenstephanensis, while no changes were observed for B. mycoides. Using Micrococcus lysodeicticus and Listeria monocytogenes as indicators lytic activity was retained by proteins of 63, 46, 38, 32 and 25 kDa and a new one of about 20 kDa in B. mycoides. Growth in the different media did not affect the autolytic pattern. NaCl abolished the activity of all the peptidoglycan hydrolases except for those of B. mycoides and B. weihenstephanensis. Lytic activity was retained in the presence of MgCl(2), MnCl(2) and EDTA and increased at basic pH. CONCLUSIONS: Bacillus cereus/B. thuringiensis/B. weihenstephanensis showed a high extent of autolysis around neutral pH, even though they presented relatively complex autolysin profiles at alkaline pH. Bacillus mycoides/B. pseudomycoides had a higher extent of autolysis at acidic pH and a simpler autolysin pattern. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on the autolytic phenotype expand the phenotypic characterization of the different species in the B. cereus group.     

Endolysosomal transport of newly-synthesized cathepsin D in a sucrose model of lysosomal storage

Newly-synthesized soluble lysosomal enzymes are transported from the trans-Golgi network to lysosomes by a mannose 6-phosphate receptor-mediated pathway. Lysosomal storage of indigestible material has been reported to perturb the biosynthesis and the fate of lysosomal hydrolases. In this study, we have focused our attention on the last steps in the transport of newly-synthesized cathepsin D to lysosomes in sucrose-treated WI-38 fibroblasts. Pulse-chase experiments indicate that, in sucrose-treated cells, cathepsin D maturation is delayed by 2 to 4 h. By subcellular fractionation, we show that newly-synthesized cathepsin D precursors transit through organelles endowed with a high sedimentation coefficient. These organelles are recovered in the dense region of a self-forming Percoll density gradient while the bulk of hydrolytic activities is redistributed to the low density region. Only later, are the precursors delivered to organelles containing the bulk of active hydrolases. There, procathepsin D is proteolytically processed into its 31 kDa-mature form. Our results suggest that when sucrose is present, the delayed maturation of procathepsin D is related to the delivery of the polypeptides into an organelle behaving in centrifugation like lysosomes but which is poorly efficient in proteolytic processing of procathepsin D. This low proteolytic activity of this organelle could be due to its poor ability to interact with hydrolase-containing structures.     

X-ray structure of a membrane-bound beta-glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii

The beta-glycosidase of the hyperthermophilic Archaeon Pyrococcus horikoshii is a membrane-bound enzyme with the preferred substrate of alkyl-beta-glycosides. In this study, the unusual structural features that confer the extreme thermostability and substrate preferences of this enzyme were investigated by X-ray crystallography and docking simulation. The enzyme was crystallized in the presence of a neutral surfactant, and the crystal structure was solved by the molecular replacement method and refined at 2.5 A. The main-chain fold of the enzyme belongs to the (betaalpha)8 barrel structure common to the Family 1 glycosyl hydrolases. The active site is located at the center of the C-termini of the barrel beta-strands. The deep pocket of the active site accepts one sugar unit, and a hydrophobic channel extending radially from there binds the nonsugar moiety of the substrate. The docking simulation for oligosaccharides and alkylglucosides indicated that alkylglucosides with a long aliphatic chain are easily accommodated in the hydrophobic channel. This sparingly soluble enzyme has a cluster of hydrophobic residues on its surface, situated at the distal end of the active site channel and surrounded by a large patch of positively charged residues. We propose that this hydrophobic region can be inserted into the membrane while the surrounding positively charged residues make favorable contacts with phosphate groups on the inner surface of the membrane. The enzyme could thus adhere to the membrane in the proximity of its glycolipid substrate.     

Regulation of Sulfoglucuronyl Glycolipid Synthesis in the Developing Rat Sciatic Nerve

Sulfoglucuronyl glycolipids (SGGLs) have been considered as target antigens in demyelinating peripheral neuropathies associated with IgM monoclonal gammopathy. The regulation of expression of SGGLs in the rat sciatic nerve during development was studied by assaying the levels of SGGLs and activities of four glycosyltransferases sequentially involved in their synthesis from lactosylceramide. The levels of SGGLs in the sciatic nerve increased with development and reached a maximum at sixty days after birth. The rate of increase in the level of SGGLs between day 5 to 20 was similar to rate of deposition of myelin in the nerve. Analysis of the activities of the glycosyltransferases showed that only lactotriosylceramide galactosyltransferase (LcOse₃Cer-GalTr) increased in parallel with the levels of SGGLs during development. The other three enzymes were not co-relative with the synthesis of SGGLs. The product of LcOse₃Cer-GalTr reaction, nLcOse₄Cer is the key intermediate for all neolactoglycolipids, particularly NeuAc2-3nLcOse₄Cer or nLM1, which is the major ganglioside (60%) of myelin in rat sciatic nerve. The results suggest that in the sciatic nerve SGGLs are mostly associated with Schwann cell myelin and their synthesis is regulated by LcOse₃Cer-GalTr, unlike in the cerebral cortex and cerebellum where SGGLs are associated with the neuronal membranes and their synthesis is regulated by lactosylceramide N-acetylglucosaminyltransferase (LcOse₂Cer-GlcNAcTr).     

General O-glycosylation of 2-furfuryl alcohol using beta-glucuronidase

beta-Glucuronidase from bovine liver is able to catalyze transfer of several carbohydrates to furfuryl alcohol, an acid-sensitive diene, with transfer yields as high as 84%. Carbohydrates that were transferred in yields of 30% or higher include gluco-, galacto-, xylo-, and fucopyranose. Small variations in the configuration of the substrate hydroxyls lead to large variations in the catalytic behavior of the enzyme in terms of both the initial reaction velocities and the final ratios of transfer-to-hydrolysis. The high transfer yields and surprising nonspecificity towards carbohydrate suggest that the enzyme may be a versatile tool for the general O-glycosylation of dienic alcohols.     

Blood group A and B glycosyltransferases synthesize A and B determinants on different acceptor polyglycosyl peptidesin vitro

The glycosylation of polyglycosyl chains from human erythrocytes by human plasma blood group A and B glycosyltransferases was studied in order to clarify why human blood group AB erythrocyte polyglycosyl peptides carry only either A or B determinants [Eur J Biochem (1981) 113:259–65].The blood group A transferase was able to add radioactiveN-acetylgalactosamine from labeled UDP-N-acetylgalactosamine to B-type erythrocytes which had been treated with -galactosidase in order to cleave the B determinant sugar from the erythrocytes. This suggests that the enzymes specified by theA andB genes utilize the same acceptor molecules on erythrocyte membranes. Polyglycosyl peptides isolated from blood group B erythrocytes acted as acceptors for blood group A glycosyltransferase and the generation of hybrid structures containing both A and B determinants was demon-strated. When blood group O polyglycosyl peptides were used as acceptors in the simultaneous presence of both blood group A and B glycosyltransferases, however, the A and B determinant sugars were found in different polyglycosyl peptides. It is suggested that the enzyme-acceptor complex does not dissociate until the final number of determinants has been added.