In vitro functional analysis and in silico structural modelling of pathogen-secreted polyglycine hydrolases.

Polyglycine hydrolases are fungal effectors composed of an N-domain with unique sequence and structure and a C-domain that resembles ß-lactamases, with serine protease activity. These secreted fungal proteins cleave Gly-Gly bonds within a polyglycine sequence in corn ChitA chitinase. The polyglycine hydrolase N-domain (PND) function is unknown. In this manuscript we provide evidence that the PND does not directly participate in ChitA cleavage. In vitro analysis of site-directed mutants in conserved residues of the PND of polyglycine hydrolase Es-cmp did not specifically impair protease activity. Furthermore, in silico structural models of three ChitA-bound polyglycine hydrolases created by High Ambiguity Driven protein-protein DOCKing (HADDOCK) did not predict significant interactions between the PND and ChitA. Together these results suggest that the PND has another function. To determine what types of PND-containing proteins exist in nature we performed a computational analysis of Foldseek-identified PND-containing proteins. The analysis showed that proteins with PNDs are present throughout biology as either single domain proteins or fused to accessory domains that are diverse but are usually proteases or kinases.

Developing the E. coli platform for efficient production of UMP-derived chemicals.

5-Methyluridine (5-MU) is a prominent intermediate for industrial synthesis of several antiviral-drugs, however, its availability over the past decades has overwhelmingly relied on chemical and enzymatic strategies. Here, we have realized efficient production of 5-MU in E. coli, for the first time, via a designer artificial pathway consisting of a two-enzyme cascade (UMP 5-methylase and phosphatase). More importantly, we have engineered the E. coli cell factory to boost 5-MU production by systematic evaluation of multiple strategies, and as a proof of concept, we have further developed an antibiotic-free fermentation strategy to realize 5-MU production (10.71 g/L) in E. coli MB229 (a ΔthyA strain). Remarkably, we have also established a versatile and robust platform with exploitation of the engineered E. coli for efficient production of diversified UMP-derived chemicals. This study paves the way for future engineering of E. coli as a synthetic biology platform for acceleratively accessing UMP-derived chemical diversities.

Steroidal glycoalkaloids contribute to anthracnose resistance in Solanum lycopersicum.

Anthracnose is a widespread plant disease caused by various species of the fungal pathogen Colletotrichum. In solanaceous plants such as tomato (Solanum lycopersicum), Colletotrichum infections exhibit a quiescent, asymptomatic state in developing fruit, followed by a transition to necrotrophic infections in ripe fruit. Through analysis of fruit tissue extracts of 95L368, a tomato breeding line that yields fruit with enhanced anthracnose resistance, we identified a role for steroidal glycoalkaloids (SGAs) in anthracnose resistance. The SGA α-tomatine and several of its derivatives accumulated at higher levels, in comparison with fruit of the susceptible tomato cultivar US28, and 95L368 fruit extracts displayed fungistatic activity against Colletotrichum. Correspondingly, ripe and unripe 95L368 fruit displayed enhanced expression of glycoalkaloid metabolic enzyme (GAME) genes, which encode key enzymes in SGA biosynthesis. Metabolomics analysis incorporating recombinant inbred lines generated from 95L368 and US28 yielded strong positive correlations between anthracnose resistance and accumulation of α-tomatine and several derivatives. Lastly, transient silencing of expression of the GAME genes GAME31 and GAME5 in anthracnose-susceptible tomato fruit yielded enhancements to anthracnose resistance. Together, our data support a role for SGAs in anthracnose defense in tomato, with a distinct SGA metabolomic profile conferring resistance to virulent Colletotrichum infections in ripe fruit.

Transcriptomes of an oceanic diatom reveal the initial and final stages of acclimation to copper deficiency.

Copper (Cu) concentration is greatly reduced in the open sea so that phytoplankton must adjust their uptake systems and acclimate to sustain growth. Acclimation to low Cu involves changes to the photosynthetic apparatus and specific biochemical reactions that use Cu, but little is known how Cu affects cellular metabolic networks. Here we report results of whole transcriptome analysis of a plastocyanin-containing diatom, Thalassiosira oceanica 1005, during its initial stages of acclimation and after long-term adaptation in Cu-deficient seawater. Gene expression profiles, used to identify Cu-regulated metabolic pathways, show downregulation of anabolic and energy-yielding reactions in Cu-limited cells. These include the light reactions of photosynthesis, carbon fixation, nitrogen assimilation and glycolysis. Reduction of these pathways is consistent with reduced growth requirements for C and N caused by slower rates of photosynthetic electron transport. Upregulation of oxidative stress defence systems persists in adapted cells, suggesting cellular damage by increased reactive oxygen species (ROS) occurs even after acclimation. Copper deficiency also alters fatty acid metabolism, possibly in response to an increase in lipid peroxidation and membrane damage driven by ROS. During the initial stages of Cu-limitation the majority of differentially regulated genes are associated with photosynthetic metabolism, highlighting the chloroplast as the primary target of low Cu availability. The results provide insights into the mechanisms of acclimation and adaptation of T. oceanica to Cu deficiency.

Light Stimulates Copper-Limited Growth of an Oceanic Diatom by Increasing Cellular Copper(II) Reduction─A Rate-Determining Step in Copper Uptake.

Uptake of Cu by Thalassiosira oceanica requires that Cu(II) is reduced to Cu(I) prior to transport across the cell membrane. The reduction step is mediated biochemically by cellular reductases active with a broad range of Cu chemical species. Here, we report on the cellular Cu(II) reduction and Cu(I) uptake of a diatom under saturating and subsaturating irradiance. An increase in growth irradiance, from 50 to 400 µmol photons m-2 s-1, increased the rate of extracellular Cu(II) reduction and steady-state Cu uptake. Under these conditions, Cu-limited cells acquired Cu more efficiently and maintained faster rates of growth than Cu-limited cells in low light. Pseudo-first-order reaction rate constants were about 70-fold faster for Cu(I) uptake than for Cu(II) reduction so that reduction was the rate-determining step in Cu acquisition. Accordingly, steady-state Cu uptake rates predicted from the reduction rate constants agreed well with measured rates of Cu uptake obtained from cultures growing at low nanomolar Cu concentrations. Transcript abundance of putative Cu(II) reductases followed a similar pattern to cupric reductase activity, increasing in Cu-limited cells and with increasing growth irradiance. The results are significant in showing Cu(II) reduction as the rate-determining step in Cu uptake: they suggest that biologically mediated Cu(II) reduction may be an important part of the Cu cycle in surface waters of the open sea.

Kilbournase, a protease-associated domain subtilase secreted by the fungal corn pathogen Stenocarpella maydis.

Subtilases are a large family of serine proteases that occur throughout biology. A small subset contain protease-associated (PA) domains that are structurally separate from but encoded within the active site. In bacteria, subtilase PA domains function to recruit specific protein substrates. Here we demonstrate that a protease secreted by the fungal corn pathogen Stenocarpella maydis, which truncates corn ChitA chitinase, is a PA domain subtilase. Protease was purified from S. maydis cultures and tryptic peptides were analyzed by LC-MS/MS. Ions were mapped to two predicted PA domain subtilases. Yeast strains were engineered to express each protease. One failed to produce recombinant protein while the other secreted protease that truncated ChitA. This protease, that we named kilbournase, was purified and characterized. It cleaved multiple peptide bonds in the amino-terminal chitin binding domain of ChitA while leaving the catalytic domain intact. Kilbournase was more active on the ChitA-B73 alloform compared to ChitA-LH82 and did not cleave AtChitIV3, a homolog from Arabidopsis thaliana, indicating a high level of specificity. Truncation of corn ChitA by kilbournase resembles truncation of human C5a by Streptococcus pyogenes ScpA, arguing that PA domain proteases in bacteria and fungi may commonly target specific host proteins.

Comparative Investigation into Formycin A and Pyrazofurin A Biosynthesis Reveals Branch Pathways for the Construction of C-Nucleoside Scaffolds.

Formycin A (FOR-A) and pyrazofurin A (PRF-A) are purine-related C-nucleoside antibiotics in which ribose and a pyrazole-derived base are linked by a C-glycosidic bond. However, the logic underlying the biosynthesis of these molecules has remained largely unexplored. Here, we report the discovery of the pathways for FOR-A and PRF-A biosynthesis from diverse actinobacteria and propose that their biosynthesis is likely initiated by a lysine N6-monooxygenase. Moreover, we show that forT and prfT (involved in FOR-A and PRF-A biosynthesis, respectively) mutants are correspondingly capable of accumulating the unexpected pyrazole-related intermediates 4-amino-3,5-dicarboxypyrazole and 3,5-dicarboxy-4-oxo-4,5-dihydropyrazole. We also decipher the enzymatic mechanism of ForT/PrfT for C-glycosidic bond formation in FOR-A/PRF-A biosynthesis. To our knowledge, ForT/PrfT represents an example of ß-RFA-P (ß-ribofuranosyl-aminobenzene 5'-phosphate) synthase-like enzymes governing C-nucleoside scaffold construction in natural product biosynthesis. These data establish a foundation for combinatorial biosynthesis of related purine nucleoside antibiotics and also open the way for target-directed genome mining of PRF-A/FOR-A-related antibiotics.IMPORTANCE FOR-A and PRF-A are C-nucleoside antibiotics known for their unusual chemical structures and remarkable biological activities. Deciphering the enzymatic mechanism for the construction of a C-nucleoside scaffold during FOR-A/PRF-A biosynthesis will not only expand the biochemical repertoire for novel enzymatic reactions but also permit target-oriented genome mining of FOR-A/PRF-A-related C-nucleoside antibiotics. Moreover, the availability of FOR-A/PRF-A biosynthetic gene clusters will pave the way for the rational generation of designer FOR-A/PRF-A derivatives with enhanced/selective bioactivity via synthetic biology strategies.

Inhibition of Erwinia amylovora by Bacillus nakamurai.

A variety of potential inhibitors were tested for the first time for the suppression of Erwinia amylovora, the causal agent of fire blight in apples and pears. Strain variability was evident in susceptibility to inhibitors among five independently isolated virulent strains of E. amylovora. However, most strains were susceptible to culture supernatants from strains of Bacillus spp., and particularly to the recently described species B. nakamurai. Minimal inhibitory concentrations (MICs) were 5-20% (vol/vol) of culture supernatant from B. nakamurai against all five strains of E. amylovora. Although Bacillus species have been previously reported to produce lipopeptide inhibitors of E. amylovora, matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS) and column chromatography indicated that the inhibitor from B. nakamurai was not a lipopeptide, but rather a novel inhibitor.

Functional CTR-type Cu(I) transporters in an oceanic diatom.

Copper concentration is so low in some remote parts of the sea it limits phytoplankton growth, but may be high enough in coastal and estuarine regions to be toxic. Acclimation to variations in Cu concentration thus requires a tightly regulated Cu transport system to help maintain Cu homeostasis. In marine species, the molecular mechanisms of Cu transport are not known. We studied Cu-responsive genes and uptake in Thalassiosira oceanica at environmentally relevant Cu concentrations varying between 0.012 and 12 900 pmol Cu' l-1 . Copper uptake rate assessed at high Cu concentration was three-fold faster in Cu-limited than in Cu-replete cells, confirming the existence of an inducible uptake pathway in this diatom. Four putative CTR-type Cu transporters (ToCTR1, ToCTR2, ToCTR3a and ToCTR3b) identified in the transcriptome shared conserved features with known high-affinity Cu(I) transporters. Expression of the CTR genes was upregulated as Cu concentration declined and cells maintained maximum rates of growth. Further decreases in Cu led to decreased growth rate and increased abundance of ToCTR3a/b. Both ToCTR3a and 3b restored growth of a Cu transport mutant, Saccharomyces cerevisiae ctr1Δctr3Δ, in Cu-deficient medium and increased the uptake rates of Cu(I) and Cu(II). Thus, ToCTR3a/3b is a high-affinity Cu(I) transporter that, in conjunction with the other ToCTRs, may enable T. oceanica to survive in Cu-deplete ocean environments and respond to natural variation in Cu availability.

Thiazolidine Peracetates: Carbohydrate Derivatives that Readily Assign cis-, trans-2,3-Monosaccharides by Gas Chromatography-Mass Spectrometry Analysis.

A novel group of carbohydrate derivatives is described that uniquely assign cis/ trans-2,3-aldose stereoisomers at low nanomolar concentrations. Aldopentoses, aldohexoses, or component aldoses from hydrolysis of polysaccharides or oligosaccharides react with cysteamine in pyridine to give quantitative formation of thiazolidines, which are subsequently peracetylated in a one-pot reaction. The nonpolar thiazolidines peracetate (TPA) derivatives are analyzed by gas chromatography and electron impact mass spectrometry (GC/EI-MS), each aldose giving rise to two TPA geometric isomers. The quantitative ratio of these diastereomers is dependent upon whether the parent monosaccharide is cis-2,3-(Rib, Lyx, Man, All, Gul, and Tal), or trans-2,3-aldose (Xyl, Ara, Glc, Gal, Ido, and Alt). TPAs generate observed EI-MS fragment ions characteristic of C1-C2 and C3-C4 bond cleavage of the parent sugars. This has been used to estimate the extent of metabolic labeling of microbial cell-wall carbohydrates, especially into the defining anomeric carbons and during aldolase / ketolase -catalyzed rearrangements.

Coordinated Biosynthesis of the Purine Nucleoside Antibiotics Aristeromycin and Coformycin in Actinomycetes.

Purine nucleoside antibiotic pairs, concomitantly produced by a single strain, are an important group of microbial natural products. Here, we report a target-directed genome mining approach to elucidate the biosynthesis of the purine nucleoside antibiotic pair aristeromycin (ARM) and coformycin (COF) in Micromonospora haikouensis DSM 45626 (a new producer for ARM and COF) and Streptomyces citricolor NBRC 13005 (a new COF producer). We also provide biochemical data that MacI and MacT function as unusual phosphorylases, catalyzing an irreversible reaction for the tailoring assembly of neplanocin A (NEP-A) and ARM. Moreover, we demonstrate that MacQ is shown to be an adenosine-specific deaminase, likely relieving the potential "excess adenosine" for producing cells. Finally, we report that MacR, an annotated IMP dehydrogenase, is actually an NADPH-dependent GMP reductase, which potentially plays a salvage role for the efficient supply of the precursor pool. Hence, these findings illustrate a fine-tuned pathway for the biosynthesis of ARM and also open the way for the rational search for purine antibiotic pairs.IMPORTANCE ARM and COF are well known for their prominent biological activities and unusual chemical structures; however, the logic of their biosynthesis has long been poorly understood. Actually, the new insights into the ARM and COF pathway will not only enrich the biochemical repertoire for interesting enzymatic reactions but may also lay a solid foundation for the combinatorial biosynthesis of this group of antibiotics via a target-directed genome mining strategy.

An ATP-Dependent Ligase with Substrate Flexibility Involved in Assembly of the Peptidyl Nucleoside Antibiotic Polyoxin.

Polyoxin (POL) is an unusual peptidyl nucleoside antibiotic, in which the peptidyl moiety and nucleoside skeleton are linked by an amide bond. However, their biosynthesis remains poorly understood. Here, we report the deciphering of PolG as an ATP-dependent ligase responsible for the assembly of POL. A polG mutant is capable of accumulating multiple intermediates, including the peptidyl moiety (carbamoylpolyoxamic acid [CPOAA]) and the nucleoside skeletons (POL-C and the previously overlooked thymine POL-C). We further demonstrate that PolG employs an ATP-dependent mechanism for amide bond formation and that the generation of the hybrid nucleoside antibiotic POL-N is also governed by PolG. Finally, we determined that the deduced ATP-binding sites are functionally essential for PolG and that they are highly conserved in a number of related ATP-dependent ligases. These insights have allowed us to propose a catalytic mechanism for the assembly of peptidyl nucleoside antibiotic via an acyl-phosphate intermediate and have opened the way for the combinatorial biosynthesis/pathway engineering of this group of nucleoside antibiotics.IMPORTANCE POL is well known for its remarkable antifungal bioactivities and unusual structural features. Actually, elucidation of the POL assembly logic not only provides the enzymatic basis for further biosynthetic understanding of related peptidyl nucleoside antibiotics but also contributes to the rational generation of more hybrid nucleoside antibiotics via synthetic biology strategy.

The influence of light on copper-limited growth of an oceanic diatom, Thalassiosira oceanica (Coscinodiscophyceae).

Thalassiosira oceanica (CCMP 1005) was grown over a range of copper concentrations at saturating and subsaturating irradiance to test the hypothesis that Cu and light were interacting essential resources. Growth was a hyperbolic function of irradiance in Cu-replete medium (263 fmol Cu' · L-1 ) with maximum rates achieved at 200 µmol photons · m-2  · s-1 . Lowering the Cu concentration at this irradiance to 30.8 fmol Cu' · L-1 decreased cellular Cu quota by 7-fold and reduced growth rate by 50%. Copper-deficient cells had significantly slower (P < 0.0001) rates of maximum, relative photosynthetic electron transport (rETRmax ) than Cu-sufficient cells, consistent with the role of Cu in photosynthesis in this diatom. In low-Cu medium (30.8 fmol Cu' · L-1 ), growth rate was best described as a positive, linear function of irradiance and reached the maximum value measured in Cu-replete cells when irradiance increased to 400 µmol photons · m-2  · s-1 . Thus, at high light, low-Cu concentration was no longer limiting to growth: Cu concentration and light interacted strongly to affect growth rate of T. oceanica (P < 0.0001). Relative ETRmax and Cu quota of cells grown at low Cu also increased at 400 µmol photons · m-2  · s-1 to levels measured in Cu-replete cells. Steady-state uptake rates of Cu-deficient and sufficient cells were light-dependent, suggesting that faster growth of T. oceanica under high light and low Cu was a result of light-stimulated Cu uptake.

A female Viking warrior confirmed by genomics.

OBJECTIVES: The objective of this study has been to confirm the sex and the affinity of an individual buried in a well-furnished warrior grave (Bj 581) in the Viking Age town of Birka, Sweden. Previously, based on the material and historical records, the male sex has been associated with the gender of the warrior and such was the case with Bj 581. An earlier osteological classification of the individual as female was considered controversial in a historical and archaeological context. A genomic confirmation of the biological sex of the individual was considered necessary to solve the issue. MATERIALS AND METHODS: Genome-wide sequence data was generated in order to confirm the biological sex, to support skeletal integrity, and to investigate the genetic relationship of the individual to ancient individuals as well as modern-day groups. Additionally, a strontium isotope analysis was conducted to highlight the mobility of the individual. RESULTS: The genomic results revealed the lack of a Y-chromosome and thus a female biological sex, and the mtDNA analyses support a single-individual origin of sampled elements. The genetic affinity is close to present-day North Europeans, and within Sweden to the southern and south-central region. Nevertheless, the Sr values are not conclusive as to whether she was of local or nonlocal origin. DISCUSSION: The identification of a female Viking warrior provides a unique insight into the Viking society, social constructions, and exceptions to the norm in the Viking time-period. The results call for caution against generalizations regarding social orders in past societies.

Identification of an iron permease, cFTR1, in cyanobacteria involved in the iron reduction/re-oxidation uptake pathway.

Cyanobacteria are globally important primary producers and abundant in many iron-limited aquatic environments. The ways in which they take up iron are largely unknown, but reduction of Fe3+ is an important step in the process. Here we report a special iron permease in Synechocystis, cFTR1, that is required for Fe3+ uptake following Fe2+ re-oxidation. The expression of cFTR1 is induced by iron starvation, and a mutant lacking the gene is abnormally sensitive to iron starvation. The cFTR1 protein localizes to the plasma membrane and contains the iron-binding motif "REXXE". Point-directed mutagenesis of the REXXE motif results in a sensitivity to Fe-deficiency. Measurements of iron (55 Fe) uptake rate show that cFTR1 takes up Fe3+ rather than Fe2+ . The function of cFTR1 in Synechocystis could be genetically complemented by the iron permease, Ftr1p, of Saccharomyces cerevisiae, that is known to transport Fe3+ produced by the oxidation of Fe2+ via a multicopper oxidase. Unlike yeast Ftr1p, cyanobacterial cFTR1 probably obtains Fe3+ primarily from the oxidation of Fe2+ by oxygen. Growth assays show that the cFTR1 is required during oxygenic, photoautotrophic growth but not when oxygen production is inhibited during photoheterotrophic growth. In cyanobacteria, iron reduction/re-oxidation uptake pathway may represent their adaptation to oxygenated environments.

Laparoscopic versus open peritoneal dialysis catheter insertion for the management of pediatric acute kidney injury.

BACKGROUND: Acute pediatric dialysis is provided by a single center in New Zealand. Most acute dialysis in our center is performed in the under 5 age group. The advantage of using peritoneal dialysis (PD) in these children is the ability to perform continuous renal replacement therapy without always requiring an ICU setting, avoiding central venous access and promoting greater cardiovascular stability. The disadvantage of PD in the acute setting includes the requirement for immediate use and the potential for early leaks due to peritoneal disruption with resulting delayed use and restricted volumes. There is a growing trend toward minimally invasive surgery and the laparoscopic method allows this. Surgeons at this center have been using a laparoscopic technique since 2005. METHODS: We performed a 10-year review of acute PD at the Starship Hospital from 2003 to 2013. Data on 102 children who met the criteria were collected. RESULTS: These 102 children had 113 acute PD catheters. The two groups were comparable in terms of age and reason for presentation. The median age of the laparoscopic group was 2 years (interquartile range [IQR] 6) and the open group was 3 years (IQR 3.2). The predominant diagnosis for both groups was hemolytic uremic syndrome (HUS) accounting for 71% of laparoscopic cases, and 72% of open cases. The incidence of infection was 0% versus 7% in the laparoscopic versus open approach. Ten percent of patients required further manipulation of the catheter after initial insertion in the laparoscopic group, compared with 11% in the open approach. Conversion to hemodialysis (HD) due to catheter-related complications was seen in 10% of laparoscopic cases and 9% of the open cases. Dialysate fluid leak was noted in 26% in the laparoscopic group compared with 11% in the open group (p = 0.08). Anesthesia time is longer in the laparoscopic group (p = 0.008). CONCLUSION: We found no significant differences in complication rates between laparoscopic and open surgical approaches regarding acute PD catheter insertion. We saw a trend in increased leakage with laparoscopic procedures and a significantly longer operative time. We concluded that the laparoscopic approach in the acute situation for emergency dialysis is safe and effective.

Production of anti-streptococcal liamocins from agricultural biomass by Aureobasidium pullulans.

Liamocins are unique heavier-than-water "oils" produced by certain strains of the fungus Aureobasidium pullulans. Liamocins have antibacterial activity with specificity for Streptococcus sp. Previous studies reported that liamocin yields were highest from strains of A. pullulans belonging to phylogenetic clades 8, 9, and 11, cultured on medium containing sucrose. In this study, 27 strains from these clades were examined for the first time for production of liamocins from agricultural biomass substrates. Liamocin yields were highest from strains in phylogenetic clade 11, and yields were higher from cultures grown on sucrose than from those grown on pretreated wheat straw. However, when supplementary enzymes (cellulase, ß-glucosidase, and xylanase) were added, liamocin production on pretreated wheat straw was equivalent to that on sucrose. Liamocins produced from wheat straw were free of the melanin contamination common in sucrose-grown cultures. Furthermore, MALDI-TOF MS analysis showed that liamocins produced from wheat straw were under-acetylated, resulting in higher proportions of the mannitol A1 and B1 species of liamocin, the latter of which has the highest biological activity against Streptococcus sp.

2-Acylamido analogues of N-acetylglucosamine prime formation of chitin oligosaccharides by yeast chitin synthase 2.

Chitin, a homopolymer of ß1,4-linked N-acetylglucosamine (GlcNAc) residues, is a key component of the cell walls of fungi and the exoskeletons of arthropods. Chitin synthases transfer GlcNAc from UDP-GlcNAc to preexisting chitin chains in reactions that are typically stimulated by free GlcNAc. The effect of GlcNAc was probed by using a yeast strain expressing a single chitin synthase, Chs2, by examining formation of chitin oligosaccharides (COs) and insoluble chitin, and by replacing GlcNAc with 2-acylamido analogues of GlcNAc. Synthesis of COs was strongly dependent on inclusion of GlcNAc in chitin synthase incubations, and N,N'-diacetylchitobiose (GlcNAc2) was the major reaction product. Formation of both COs and insoluble chitin was also stimulated by GlcNAc2 and by N-propanoyl-, N-butanoyl-, and N-glycolylglucosamine. MALDI analyses of the COs made in the presence of 2-acylamido analogues of GlcNAc showed they that contained a single GlcNAc analogue and one or more additional GlcNAc residues. These results indicate that Chs2 can use certain 2-acylamido analogues of GlcNAc, and likely free GlcNAc and GlcNAc2 as well, as GlcNAc acceptors in a UDP-GlcNAc-dependent glycosyltransfer reaction. Further, formation of modified disaccharides indicates that CSs can transfer single GlcNAc residues.

Nickel-Catalyzed Proton-Deuterium Exchange (HDX) Procedures for Glycosidic Linkage Analysis of Complex Carbohydrates.

The structural analysis of complex carbohydrates typically requires the assignment of three parameters: monosaccharide composition, the position of glycosidic linkages between monosaccharides, and the position and nature of noncarbohydrate substituents. The glycosidic linkage positions are often determined by permethylation analysis, but this can be complicated by high viscosity or poor solubility, resulting in under-methylation. This is a drawback because an under-methylated position may be misinterpreted as the erroneous site of a linkage or substituent. Here, we describe an alternative approach to linkage analysis that makes use of a nonreversible deuterium exchange of C-H protons on the carbohydrate backbone. The exchange reaction is conducted in deuterated water catalyzed by Raney nickel, and results in the selective exchange of C-H protons adjacent to free hydroxyl groups. Hence, the position of the residual C-H protons is indicative of the position of glycosidic linkages or other substituents and can be readily assigned by heteronuclear single quantum coherence-nuclear magnetic resonance (HSQC-NMR) or, following suitable derivatization, by gas chromatography-mass spectroscopy (GC/MS) analysis. Moreover, because the only changes to the parent sugar are proton/deuterium exchanges, the composition and linkage analysis can be determined in a single step.

Polyglycine hydrolases secreted by Pleosporineae fungi that target the linker region of plant class IV chitinases.

Cmps (chitinase-modifying proteins) are fungal proteases that truncate plant class IV chitinases by cleaving near their N-termini. We previously described Fv-cmp, a fungalysin protease that cleaves a conserved glycine-cysteine bond within the hevein domain. In the present paper we describe a new type of cmp, polyglycine hydrolases, as proteases that selectively cleave glycine-glycine peptide bonds within the polyglycine linker of plant class IV chitinases. Polyglycine hydrolases were purified from Cochliobolus carbonum (syn. Bipolaris zeicola; Bz-cmp) and Epicoccum sorghi (syn. Phoma sorghina; Es-cmp) and were shown to cleave three different maize class IV chitinase substrates. The proteolytic cleavage sites were assessed by SDS/PAGE and MALDI-TOF-MS and indicated the cleavage of multiple peptide bonds within the polyglycine linker regions. Site-directed mutagenesis was used to produce mutants of maize ChitB chitinase in which two serine residues in its linker were systematically modified to glycine. Serine to glycine changes in the ChitB linker resulted in higher susceptibility to truncation by Bz-cmp and altered substrate specificity for Bz-cmp and Es-cmp, such that different glycine-glycine peptide bonds were cleaved. Removal of the hevein domain led to loss of Es-cmp activity, indicating that interactions outside of the active site are important for recognition. Our findings demonstrate that plant class IV chitinases with polyglycine linkers are targeted for truncation by selective polyglycine hydrolases that are secreted by plant pathogenic fungi. This novel proteolysis of polyglycine motifs is previously unreported, but the specificity is similar to that of bacterial lysostaphin proteases, which cleave pentaglycine cross-links from peptidoglycan.