Molecular basis for substrate recognition and septum cleavage by AtlA, the major N-acetylglucosaminidase of Enterococcus faecalis.

The cleavage of septal peptidoglycan at the end of cell division facilitates the separation of the two daughter cells. The hydrolases involved in this process (called autolysins) are potentially lethal enzymes that can cause cell death; their activity, therefore, must be tightly controlled during cell growth. In Enterococcus faecalis, the N-acetylglucosaminidase AtlA plays a predominant role in cell separation. atlA mutants form long cell chains and are significantly less virulent in the zebrafish model of infection. The attenuated virulence of atlA mutants is underpinned by a limited dissemination of bacterial chains in the host organism and a more efficient uptake by phagocytes that clear the infection. AtlA has structural homologs in other important pathogens, such as Listeria monocytogenes and Salmonella typhimurium, and therefore represents an attractive model to design new inhibitors of bacterial pathogenesis. Here, we provide a 1.45 Å crystal structure of the E. faecalis AtlA catalytic domain that reveals a closed conformation of a conserved ß-hairpin and a complex network of hydrogen bonds that bring two catalytic residues to the ideal distance for an inverting mechanism. Based on the model of the AtlA-substrate complex, we identify key residues critical for substrate recognition and septum cleavage during bacterial growth. We propose that this work will provide useful information for the rational design of specific inhibitors targeting this enterococcal virulence factor and its orthologs in other pathogens.

Retrospective and integrative analyses of molecular characteristics and their specific imaging parameters in pediatric grade 1 gliomas.

BACKGROUND: Pediatric low-grade gliomas (PLGG) are the most common brain tumors diagnosed during childhood and represent a heterogeneous group associating variable molecular abnormalities. To go further and develop specific statistical patterns between tumor molecular background, imaging features, and patient outcome, a retrospective study was performed in a group of non-neurofibromatosis type 1 (non-NF1) grade 1 PLGGs. PATIENTS AND METHODS: Seventy-eight children, followed from 2004 to 2017, were retrospectively reported. In this population, we analyzed radiological and molecular parameters. Their therapeutic management comprised surgery or surgery plus chemotherapies. RESULTS: Considering all 78 patients, 59 had only a surgical removal and 19 patients were treated with postoperative chemotherapy. Twelve progressions were reported in the partially resected and chemotherapeutic groups, whereas four deaths occurred only in the highly treated patients. As expected, in the global cohort, PLGG with BRAF p.V600E and/or CDKN2A loss exhibited poor outcomes and we evidenced significant associations between those molecular characteristics and their imaging presentation. In the chemo-treated patients, when associating initial and 6-month magnetic resonance imaging (MRI) parameters to the molecular features, the good risk situations were significantly linked to the presence of a large tumor cyst at diagnosis and the appearance during treatment of a higher cystic proportion that we called cystic conversion. CONCLUSION: So, additionally to the presence of BRAF p.V600E or CDKN2A deletion in grade 1 PLGGs, the absence on diagnostic MRI of cystic parts and/or cystic conversion at 6-month chemotherapy were significantly linked to a worst prognosis and response to treatment. These imaging features should be considered as prognostic markers in future PLGG studies.

Pictorial review of the postmortem computed tomography in neonaticide cases.

Neonaticide is defined by the deliberate killing or homicide of a child within 24 h of its birth. In this context, three fundamental questions are generally asked of the forensic pathologist: what is the cause of death of the neonate? Was the child viable (i.e., what is the gestational age of the neonate)? Finally, was the neonate stillborn or liveborn?Postmortem imaging can help answer these questions by conducting (1) a complete lesional analysis of the body and the placenta, (2) an estimation of the gestational age by measuring the lengths of the diaphyseal long bones, and (3) an analysis of the aeration of the lungs and intestines. Using the details of 18 cases, we illustrate aspects of neonaticide cases in postmortem computed tomography (PMCT), offering detailed examples of notable postmortem changes and abnormalities, especially in the analysis of the pulmonary parenchyma. This article presents a useful iconography for the radiologist confronted with this rare yet complex forensic situation.

Structural insights into a family 39 glycoside hydrolase from the gut symbiont Bacteroides cellulosilyticus WH2.

Bacteria from the human gut are equipped with an arsenal of carbohydrate-active enzymes that degrade dietary and host-derived glycans. In this study, we present the 2.5Å resolution crystal structure of a member (GH39wh2) from the human gut bacteria Bacteroides cellulosilyticus WH2 representative of a new subgroup within family GH39. Together with 6 other GHs, GH39wh2 belongs to a polysaccharide utilization locus (PUL) that could be involved in detecting, binding and hydrolysing a specific carbohydrate species from the intestinal tract. GH39wh2 shares a similar architecture as other members of family GH39 dominated by a typical (ß/α)8-barrel fold harboring the catalytic residues and decorated by ß-sandwich accessory domains. The GH39wh2 structure unveils an atypical shallow groove rather than a deep pocket due to drastic rearrangements in surface loops surrounding the catalytic interface. These structural adaptations seem to favour recognition of large branched substrates and may explain the lack of activity of GH39wh2 toward small xylose-based and other typical substrates from GH39 members, emphasizing the molecular diversity within the GH39 family. A phylogenetic analysis of the entire GH39 family assigns GH39wh2 as a new subgroup, consistent with the extensive remodelling of the active site region that may confer new substrate specificity toward a complex glycan chain.

Structural and functional characterization of the Clostridium perfringens N-acetylmannosamine-6-phosphate 2-epimerase essential for the sialic acid salvage pathway.

Pathogenic bacteria are endowed with an arsenal of specialized enzymes to convert nutrient compounds from their cell hosts. The essential N-acetylmannosamine-6-phosphate 2-epimerase (NanE) belongs to a convergent glycolytic pathway for utilization of the three amino sugars, GlcNAc, ManNAc, and sialic acid. The crystal structure of ligand-free NanE from Clostridium perfringens reveals a modified triose-phosphate isomerase (ß/α)8 barrel in which a stable dimer is formed by exchanging the C-terminal helix. By retaining catalytic activity in the crystalline state, the structure of the enzyme bound to the GlcNAc-6P product identifies the topology of the active site pocket and points to invariant residues Lys(66) as a putative single catalyst, supported by the structure of the catalytically inactive K66A mutant in complex with substrate ManNAc-6P. (1)H NMR-based time course assays of native NanE and mutated variants demonstrate the essential role of Lys(66) for the epimerization reaction with participation of neighboring Arg(43), Asp(126), and Glu(180) residues. These findings unveil a one-base catalytic mechanism of C2 deprotonation/reprotonation via an enolate intermediate and provide the structural basis for the development of new antimicrobial agents against this family of bacterial 2-epimerases.

Structural and biochemical characterization of the ß-N-acetylglucosaminidase from Thermotoga maritima: toward rationalization of mechanistic knowledge in the GH73 family.

Members of the GH73 glycosidase family cleave the ß-1,4-glycosidic bond between the N-acetylglucosaminyl (GlcNAc) and N-acetylmuramyl (MurNAc) moieties in bacterial peptidoglycan. A catalytic mechanism has been proposed for members FlgJ, Auto, AcmA and Atl(WM) and the structural analysis of FlgJ and Auto revealed a conserved α/ß fold reminiscent of the distantly related GH23 lysozyme. Comparison of the active site residues reveals variability in the nature of the catalytic general base suggesting two distinct catalytic mechanisms: an inverting mechanism involving two distant glutamate residues and a substrate-assisted mechanism involving anchimeric assistance by the C2-acetamido group of the GlcNAc moiety. Herein, we present the biochemical characterization and crystal structure of TM0633 from the hyperthermophilic bacterium Thermotoga maritima. TM0633 adopts the α/ß fold of the family and displays ß-N-acetylglucosaminidase activity on intact peptidoglycan sacculi. Site-directed mutagenesis identifies Glu34, Glu65 and Tyr118 as important residues for catalysis. A thorough bioinformatic analysis of the GH73 sequences identified five phylogenetic clusters. TM0633, FlgJ and Auto belong to a group of three clusters that conserve two carboxylate residues involved in a classical inverting acid-base mechanism. Members of the other two clusters lack a conserved catalytic general base supporting a substrate-assisted mechanism. Molecular modeling of representative members from each cluster suggests that variability in length of the ß-hairpin region above the active site confers ligand-binding specificity and modulates the catalytic mechanisms within the GH73 family.

Medication errors involving intravenous paracetamol in children: experience from enquiries to the National Poisons Information Service.

INTRODUCTION: Children are at higher risk of medication errors due to the complexity of drug prescribing and administration in this patient group. Intravenous (IV) paracetamol overdose differs from overdose by ingestion as there is no enteral absorptive buffering. We provide the first national UK data focusing on paediatric IV paracetamol poisoning. METHODS: All telephone enquiries to the National Poisons Information Service between 2008 and 2021 regarding children less than 18 years old in the UK concerning IV paracetamol overdose were extracted from the UK Poisons Information Database (UKPID). Data were analysed using descriptive statistics. RESULTS: Enquiries were made concerning 266 children, mostly involving children under the age of 1 year (n=145; 54.5%). Acute and staggered overdoses were the most frequent types of exposure. Common error themes included 10-fold overdose in 45 cases (16.9%) and inadvertent concomitant oral and IV dosing in 64 cases (24.1%). A high proportion of cases were asymptomatic (87.1%), with many calls regarding overdoses below the treatable dose of 60 mg/kg (41.4%). Treatment with the antidote acetylcysteine was advised in 113 cases (42.5%). CONCLUSIONS: Inadvertent IV paracetamol overdose appears to occur more frequently in young children. A significant proportion were calculation errors which were often 10-fold errors. While these errors have the potential for causing serious harm, thankfully most cases were asymptomatic. Errors with IV paracetamol might be reduced by electronic prescribing support systems, better communication regarding administration and consideration of whether other routes are more appropriate.

Insights into the atypical autokinase activity of the Pseudomonas aeruginosa GacS histidine kinase and its interaction with RetS.

Virulence in Pseudomonas aeruginosa (PA) depends on complex regulatory networks, involving phosphorelay systems based on two-component systems (TCSs). The GacS/GacA TCS is a master regulator of biofilm formation, swarming motility, and virulence. GacS is a membrane-associated unorthodox histidine kinase (HK) whose phosphorelay signaling pathway is inhibited by the RetS hybrid HK. Here we provide structural and functional insights into the interaction of GacS with RetS. The structure of the GacS-HAMP-H1 cytoplasmic regions reveals an unusually elongated homodimer marked by a 135 Å long helical bundle formed by the HAMP, the signaling helix (S helix) and the DHp subdomain. The HAMP and S helix regions are essential for GacS signaling and contribute to the GacS/RetS binding interface. The structure of the GacS D1 domain together with the discovery of an unidentified functional ND domain, essential for GacS full autokinase activity, unveils signature motifs in GacS required for its atypical autokinase mechanism.

Together Intra-Tumor Hypoxia and Macrophagic Immunity Are Driven Worst Outcome in Pediatric High-Grade Osteosarcomas.

BACKGROUND: Osteosarcomas (OTS) represent the most common primary bone cancer diagnosed in adolescents and young adults. Despite remarkable advances, there are no objective molecular or imaging markers able to predict an OTS outcome at diagnosis. Focusing on biomarkers contributing broadly to treatment resistance, we examine the interplay between the tumor-associated macrophages and intra-tumor hypoxia. METHODS: Radiological and immunohistochemical (IHC) data were correlated with the outcome in a retrospective and monocentric cohort of 30 pediatric OTS. We studied hypoxic (pS6, phospho-mTor, HIF-1α and carbonic anhydrase IX (CAIX)) and macrophagic (CD68 and CD163) biomarkers. RESULTS: The imaging analyses were based on MRI manual volumetric measures on axial post-contrast T1 weighted images, where, for each tumor, we determined the necrotic volume and its ratio to the entire tumor volume. When they were above 50 cm3 and 20%, respectively, they correlated with a worse overall survival (p = 0.0072 and p = 0.0136, respectively) and event-free survival (p = 0.0059 and p = 0.0143, respectively). IHC assessments enable a significant statistical link between HIF-1α/CAIX hyper-expressions, CD68+ cells and a worse outcome, whereas activation of mTor pathway was linked to a better survival rate and CD163+ cells. CONCLUSIONS: This study evidenced the links between hypoxia and immunity in OTS, as their poor outcome may be related to a larger necrotic volume on diagnostic MRI and, in biopsies, to a specific IHC profile.

Crystal structures of a family 8 polysaccharide lyase reveal open and highly occluded substrate-binding cleft conformations.

Bacterial enzymatic degradation of glycosaminoglycans such as hyaluronan and chondroitin is facilitated by polysaccharide lyases. Family 8 polysaccharide lyase (PL8) enzymes contain at least two domains: one predominantly composed of α-helices, the α-domain, and another predominantly composed of ß-sheets, the ß-domain. Simulation flexibility analyses indicate that processive exolytic cleavage of hyaluronan, by PL8 hyaluronate lyases, is likely to involve an interdomain shift, resulting in the opening/closing of the substrate-binding cleft between the α- and ß-domains, facilitating substrate translocation. Here, the Streptomyces coelicolor A3(2) PL8 enzyme was recombinantly expressed in and purified from Escherichia coli and biochemically characterized as a hyaluronate lyase. By using X-ray crystallography its structure was solved in complex with hyaluronan and chondroitin disaccharides. These findings show key catalytic interactions made by the different substrates, and on comparison with all other PL8 structures reveals that the substrate-binding cleft of the S. coelicolor enzyme is highly occluded. A third structure of the enzyme, harboring a mutation of the catalytic tyrosine, created via site-directed mutagenesis, interestingly revealed an interdomain shift that resulted in the opening of the substrate-binding cleft. These results add further support to the proposed processive mechanism of action of PL8 hyaluronate lyases and may indicate that the mechanism of action is likely to be universally used by PL8 hyaluronate lyases.

Distinct oligomeric forms of the Pseudomonas aeruginosa RetS sensor domain modulate accessibility to the ligand binding site.

Bacterial two-component regulatory systems (TCSs) sense environmental stimuli to adapt the lifestyle of microbial populations. For many TCSs the stimulus is a ligand of unknown chemical nature. Pseudomonas aeruginosa utilizes the closely related RetS and LadS sensor kinases to switch between acute and chronic infections. These sensor proteins antagonistically mediate biofilm formation through communication with a central TCS, GacA/GacS. Recently, it was shown that RetS modulates the GacS sensor activity by forming RetS/GacS heterodimers. LadS and RetS are hybrid sensors with a signalling domain consisting of a 7-transmembrane (7TMR) region and a periplasmic sensor domain (diverse intracellular signalling module extracellular 2, DISMED2). The 2.65 A resolution crystal structure of RetS DISMED2, called RetSp, reveals three distinct oligomeric states capable of domain swapping. The RetSp structure also displays two putative ligand binding sites. One is equivalent to the analogous site in the structurally-related carbohydrate binding module (CBM) but the second site is located at a dimer interface. These observations highlight the modular architecture and assembly of the RetSp fold and give clues on how homodimerization of RetS could be modulated upon ligand binding to control formation of a RetS/GacS heterodimer. Modelling the DISMED2 of LadS reveals conservation of only one ligand binding site, suggesting a distinct mechanism underlying the activity of this sensor kinase.

Biochemical characterization of a glycosyltransferase Gtf3 from Mycobacterium smegmatis: a case study of improved protein solubilization.

Glycosyltransferases (GTs) are widely present in several organisms. These enzymes specifically transfer sugar moieties to a range of substrates. The processes of bacterial glycosylation of the cell wall and their relations with host-pathogen interactions have been studied extensively, yet the majority of mycobacterial GTs involved in the cell wall synthesis remain poorly characterized. Glycopeptidolipids (GPLs) are major class of glycolipids present on the cell wall of various mycobacterial species. They play an important role in drug resistance and host-pathogen interaction virulence. Gtf3 enzyme performs a key step in the biosynthesis of triglycosylated GPLs. Here, we describe a general procedure to achieve expression, purification, and crystallization of recombinant protein Gtf3 from Mycobacterium smegmatis using an E. coli expression system. We reported also a combined bioinformatics and biochemical methods to predict aggregation propensity and improve protein solubilization of recombinant Gtf3. NVoy, a carbohydrate-based polymer reagent, was added to prevent protein aggregation by binding to hydrophobic protein surfaces of Gtf3. Using intrinsic tryptophan fluorescence quenching experiments, we also demonstrated that Gtf3-NVoy enzyme interacted with TDP and UDP nucleotide ligands. This case report proposes useful tools for the study of other glycosyltransferases which are rather difficult to characterize and crystallize.

SFCE-RAPIRI Phase I Study of Rapamycin Plus Irinotecan: A New Way to Target Intra-Tumor Hypoxia in Pediatric Refractory Cancers.

Hypoxic environment is a prognostic factor linked in pediatric cancers to a worse outcome, favoring tumor progression and resistance to treatments. The activation of mechanistic Target Of Rapamycin (mTor)/hypoxia inducible factor (HIF)-1 pathway can be targeted by rapamycin and irinotecan, respectively. Therefore, we designed a phase I trial associating both drugs in pediatric refractory/relapsing solid tumors. Patients were enrolled according to a 3 + 3 escalation design with ten levels, aiming to determine the MTD (maximum tolerated dose) of rapamycin plus irinotecan. Rapamycin was administered orally once daily in a 28-day cycle (1 to 2.5 mg/m2/day), associating biweekly intravenous irinotecan (125 to 240 mg/m2/dose). Toxicities, pharmacokinetics, efficacy analyses, and pharmacodynamics were evaluated. Forty-two patients, aged from 2 to 18 years, were included. No MTD was reached. Adverse events were mild to moderate. Only rapamycin doses of 1.5 mg/m2/day reached over time clinically active plasma concentrations. Tumor responses and prolonged stable disease were associated with a mean irinotecan area under the curve of more than 400 min.mg/L. Fourteen out of 31 (45.1%) patients had a non-progressive disease at 8 weeks. Most of them were sarcomas and brain tumors. For the phase II trial, we can then propose biweekly 125 mg/m2 irinotecan dose with a pharmacokinetic (PK) follow-up and a rapamycin dose of 1.5 mg/m2/day, reaching a blood concentration above 10 g/L.

Safety, efficacy and predictive factors of efficacy of a single intra-articular injection of non-animal-stabilized-hyaluronic-acid in the hip joint: results of a standardized follow-up of patients treated for hip osteoarthritis in daily practice.

AIM: To evaluate, in daily clinical practice, the efficacy and tolerability of a single intra-articular injection of non-animal-stabilized hyaluronic acid (NASHA) in patients treated for symptomatic hip OA (HOA). METHODS: Standardized follow-up (FU). PATIENTS: forty patients suffering from HOA treated by a single intra-articular injection of NASHA in the painful hip under fluoroscopy. EVALUATION: patient global assessment (PGA) and walking pain (WP) on a 100 mm visual analogue scale, WOMAC index, Lequesne index at each visit. STATISTICS: last observation carried forward. Treatment efficacy was assessed using OMERACT-OARSI response criteria, minimal clinically important improvement (MCII), patient acceptable symptom state (PASS) obtained from PGA, WOMAC and WP. Predictive factors of efficacy were also studied. RESULTS: Efficacy evaluation: 34 patients were assessable (mean FU 159 days). All clinical variables (WP, PGA, WOMAC, Lequesne index) decreased significantly between baseline and last evaluation. Twenty-two patients (71%) were classified OMERACT-OARSI responders, 25 subjects (75.8%) were classified PASS+, and 19 (61.3%) fulfilled criteria for MCII. Out of clinical and radiological variables only Lequesne index (p = 0.04) and WOMAC (p = 0.04) at baseline were found to be predictive of treatment efficacy. Safety evaluation: the treatment was well tolerated. There were no severe adverse events related to the treatment or to the procedure. However 15 of the 28 assessable patients experienced transient increase of pain in the target hip during the first week after injection. CONCLUSION: Viscosupplementation of the hip with NASHA is easily feasible in daily clinical practice, safe and well tolerated despite a frequent increase of pain the days following injection. Prospective controlled trials are needed to confirm these data and to evaluate both safety and efficacy of a second course of treatment.

NMR assignments of the GacS histidine-kinase periplasmic detection domain from Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is a highly adaptable opportunistic pathogen. It can infect vulnerable patients such as those with cystic fibrosis or hospitalized in intensive care units where it is responsible for both acute and chronic infection. The switch between these infections is controlled by a complex regulatory system involving the central GacS/GacA two-component system that activates the production of two small non-coding RNAs. GacS is a histidine kinase harboring one periplasmic detection domain, two inner-membrane helices and three H1/D1/H2 cytoplasmic domains. By detecting a yet unknown signal, the GacS histidine-kinase periplasmic detection domain (GacSp) is predicted to play a key role in activating the GacS/GacA pathway. Here, we present the chemical shift assignment of 96 % of backbone atoms (HN, N, C, Cα, Cß and Hα), 88 % aliphatic hydrogen atoms and 90 % of aliphatic carbon atoms of this domain. The NMR-chemical shift data, on the basis of Talos server secondary structure predictions, reveal that GacSp consists of 3 ß-strands, 3 α-helices and a major loop devoid of secondary structures.

Structural and functional insights into the periplasmic detector domain of the GacS histidine kinase controlling biofilm formation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogenic bacterium responsible for both acute and chronic infections and has developed resistance mechanisms due to its ability to promote biofilm formation and evade host adaptive immune responses. Here, we investigate the functional role of the periplasmic detector domain (GacSPD) from the membrane-bound GacS histidine kinase, which is one of the key players for biofilm formation and coordination of bacterial lifestyles. A gacS mutant devoid of the periplasmic detector domain is severely defective in biofilm formation. Functional assays indicate that this effect is accompanied by concomitant changes in the expression of the two RsmY/Z small RNAs that control activation of GacA-regulated genes. The solution NMR structure of GacSPD reveals a distinct PDC/PAS α/ß fold characterized by a three-stranded ß-sheet flanked by α-helices and an atypical major loop. Point mutations in a putative ligand binding pocket lined by positively-charged residues originating primarily from the major loop impaired biofilm formation. These results demonstrate the functional role of GacSPD, evidence critical residues involved in GacS/GacA signal transduction system that regulates biofilm formation, and document the evolutionary diversity of the PDC/PAS domain fold in bacteria.

Postpneumonectomy Compression of the Mitral Annulus: Rare Vascular Complication in Sportive Patient.

Numerous postpneumonectomy complications exist. We present a rare clinical case of postpneumonectomy exertional dyspnea revealing compression of the mitral annulus by the descending aorta. The patient was 42-year-old former smoker with pulmonary emphysema. He has been operated on, in 2012 (i.e., right pneumonectomy). Before the surgery, the patient was a recreational runner. However, after some months, it was difficult for the patient to resume running. Cardiopulmonary exercise testing indicated moderate exercise intolerance with important oxygen desaturation. More interestingly, a decrease of low oxygen pulse was noticed from the first ventilatory threshold with no electrical modification on the electrocardiogram. This decrease was indicative of a decline in stroke volume. The thoracic scan revealed a right pneumonectomy pocket with a liquid abnormal content. Moreover, the mediastinum had shifted toward the pneumonectomy space and the left lung was distended and emphysematous. Echocardiography revealed a major change in the mediastinal anatomy. The mitral annulus was observed to be compressed by the rear wall of the descending aorta. The diagnosis of postpneumonectomy syndrome or platypnea-orthodeoxia syndrome was ruled out in this patient. Mitral annular compression by the descending aorta is rare complication, which must be researched in patients with postpneumonectomy exertional dyspnea.

Multifunctional xylooligosaccharide/cephalosporin C deacetylase revealed by the hexameric structure of the Bacillus subtilis enzyme at 1.9A resolution.

Esterases and deacetylases active on carbohydrate ligands have been classified into 14 families based upon amino acid sequence similarities. Enzymes from carbohydrate esterase family seven (CE-7) are unusual in that they display activity towards both acetylated xylooligosaccharides and the antibiotic, cephalosporin C. The 1.9A structure of the multifunctional CE-7 esterase (hereinafter CAH) from Bacillus subtilis 168 reveals a classical alpha/beta hydrolase fold encased within a 32 hexamer. This is the first example of a hexameric alpha/beta hydrolase and is further evidence of the versatility of this particular fold, which is used in a wide variety of biological contexts. A narrow entrance tunnel leads to the centre of the molecule, where the six active-centre catalytic triads point towards the tunnel interior and thus are sequestered away from cytoplasmic contents. By analogy to self-compartmentalising proteases, the tunnel entrance may function to hinder access of large substrates to the poly-specific active centre. This would explain the observation that the enzyme is active on a variety of small, acetylated molecules. The structure of an active site mutant in complex with the reaction product, acetate, reveals details of the putative oxyanion binding site, and suggests that substrates bind predominantly through non-specific contacts with protein hydrophobic residues. Protein residues involved in catalysis are tethered by interactions with protein excursions from the canonical alpha/beta hydrolase fold. These excursions also mediate quaternary structure maintenance, so it would appear that catalytic competence is only achieved on protein multimerisation. We suggest that the acetyl xylan esterase (EC 3.1.1.72) and cephalosporin C deacetylase (EC 3.1.1.41) enzymes of the CE-7 family represent a single class of proteins with a multifunctional deacetylase activity against a range of small substrates.

Identification of the nucleophile catalytic residue of GH51 α-L-arabinofuranosidase from Pleurotus ostreatus.

In this study, the recombinant α-L-arabinofuranosidase from the fungus Pleurotus ostreatus (rPoAbf) was subjected to site-directed mutagenesis in order to identify the catalytic nucleophile residue. Based on bioinformatics and homology modelling analyses, E449 was revealed to be the potential nucleophilic residue. Thus, the mutant E449G of PoAbf was recombinantly expressed in Pichia pastoris and its recombinant expression level and reactivity were investigated in comparison to the wild-type. The design of a suitable set of hydrolysis experiments in the presence or absence of alcoholic arabinosyl acceptors and/or formate salts allowed to unambiguously identify the residue E449 as the nucleophile residue involved in the retaining mechanism of this GH51 arabinofuranosidase. (1)H NMR analysis was applied for the identification of the products and the assignement of their anomeric configuration.