The Retaining Pse5Ac7Ac Pseudaminyltransferase KpsS1 Defines a Previously Unreported glycosyltransferase family (GT118).

Cell surface sugar 5,7-diacetyl pseudaminic acid (Pse5Ac7Ac) is a bacterial analogue of the ubiquitous sialic acid, Neu5Ac, and contributes to the virulence of a number of multidrug resistant bacteria, including ESKAPE pathogens Pseudomonas aeruginosa, and Acinetobacter baumannii. Despite its discovery in the surface glycans of bacteria over thirty years ago, to date no glycosyltransferase enzymes (GTs) dedicated to the synthesis of a pseudaminic acid glycosidic linkage have been unequivocally characterised in vitro. Herein we demonstrate that A. baumannii KpsS1 is a dedicated pseudaminyltransferase enzyme (PseT) which constructs a Pse5Ac7Ac-α(2,6)-Glcp linkage, and proceeds with retention of anomeric configuration. We utilise this PseT activity in tandem with the biosynthetic enzymes required for CMP-Pse5Ac7Ac assembly, in a two-pot, seven enzyme synthesis of an α-linked Pse5Ac7Ac glycoside. Due to its unique activity and protein sequence, we also assign KpsS1 as the prototypical member of a previously unreported GT family (GT118).

Reverse thiophosphorylase activity of a glycoside phosphorylase in the synthesis of an unnatural Manß1,4GlcNAc library.

ß-Mannosides are ubiquitous in nature, with diverse roles in many biological processes. Notably, Manß1,4GlcNAc a constituent of the core N-glycan in eukaryotes was recently identified as an immune activator, highlighting its potential for use in immunotherapy. Despite their biological significance, the synthesis of ß-mannosidic linkages remains one of the major challenges in glycoscience. Here we present a chemoenzymatic strategy that affords a series of novel unnatural Manß1,4GlcNAc analogues using the ß-1,4-d-mannosyl-N-acetyl-d-glucosamine phosphorylase, BT1033. We show that the presence of fluorine in the GlcNAc acceptor facilitates the formation of longer ß-mannan-like glycans. We also pioneer a "reverse thiophosphorylase" enzymatic activity, favouring the synthesis of longer glycans by catalysing the formation of a phosphorolysis-stable thioglycoside linkage, an approach that may be generally applicable to other phosphorylases.

Ultrafast 2D-IR spectroscopy of [NiFe] hydrogenase from E. coli reveals the role of the protein scaffold in controlling the active site environment.

Ultrafast two-dimensional infrared (2D-IR) spectroscopy of Escherichia coli Hyd-1 (EcHyd-1) reveals the structural and dynamic influence of the protein scaffold on the Fe(CO)(CN)2 unit of the active site. Measurements on as-isolated EcHyd-1 probed a mixture of active site states including two, which we assign to Nir-SI/II, that have not been previously observed in the E. coli enzyme. Explicit assignment of carbonyl (CO) and cyanide (CN) stretching bands to each state is enabled by 2D-IR. Energies of vibrational levels up to and including two-quantum vibrationally excited states of the CO and CN modes have been determined along with the associated vibrational relaxation dynamics. The carbonyl stretching mode potential is well described by a Morse function and couples weakly to the cyanide stretching vibrations. In contrast, the two CN stretching modes exhibit extremely strong coupling, leading to the observation of formally forbidden vibrational transitions in the 2D-IR spectra. We show that the vibrational relaxation times and structural dynamics of the CO and CN ligand stretching modes of the enzyme active site differ markedly from those of a model compound K[CpFe(CO)(CN)2] in aqueous solution and conclude that the protein scaffold creates a unique biomolecular environment for the NiFe site that cannot be represented by analogy to simple models of solvation.

A Tale of Two Bioconjugations: pH Controlled Divergent Reactivity of Protein α-oxo-Aldehydes in Competing α-oxo-Mannich and Catalyst-Free Aldol Ligations.

Site-selective chemical methods for protein bioconjugation have revolutionized the fields of cell and chemical biology through the development of novel protein/enzyme probes bearing fluorescent, spectroscopic, or even toxic cargos. Herein, we report two new methods for the bioconjugation of α-oxo aldehyde handles within proteins using small molecule aniline and/or phenol probes. The "α-oxo-Mannich" and "catalyst-free aldol" ligations both compete for the electrophilic α-oxo aldehyde, which displays pH divergent reactivity proceeding through the "Mannich" pathway at acidic pH to afford bifunctionalized bioconjugates, and the "catalyst-free aldol" pathway at neutral pH to afford monofunctionalized bioconjugates. We explore the substrate scope and utility of both of these bioconjugations in the construction of neoglycoproteins, in the process formulating a mechanistic rationale for how both pathways intersect with each other at different reaction pH's.

Current concepts in the rehabilitation of rotator cuff related disorders.

Rotator cuff related disorders (RCRD) are common. Exercise-based rehabilitation can improve outcomes, yet uncertainty exists regarding the characteristics of these exercises. This scoping review paper summarises the key characteristics of the exercise-based rehabilitation of rotator cuff related disorders (RCRD). An iterative search process was used to capture the breadth of current evidence and a narrative summary of the data was produced. 57 papers were included. Disagreement around terminology, diagnostic standards, and outcome measures limits the comparison of the data. Rehabilitation should utilise a biopsychosocial approach, be person-centred and foster self-efficacy. Biomedically framed beliefs can create barriers to rehabilitation. Pain drivers in RCRSD are unclear, as is the influence of pain during exercise on outcomes. Expectations and preferences around pain levels should be discussed to allow the co-creation of a programme that is tolerated and therefore engaged with. The optimal parameters of exercise-based rehabilitation remain unclear; however, programmes should be individualised and progressive, with a minimum duration of 12 weeks. Supervised or home-based exercises are equally effective. Following rotator cuff repair, rehabilitation should be milestone-driven and individualised; communication across the MDT is essential. For individuals with massive rotator cuff tears, the anterior deltoid programme is a useful starting point and should be supplemented by functional rehabilitation, exercises to optimise any remaining cuff and the rest of the kinetic chain. In conclusion, exercise-based rehabilitation improves outcomes for individuals with a range of RCRD. The optimal parameters of these exercises remain unclear. Variation exists across current physiotherapy practice and post-operative rehabilitation protocols, reflecting the wide-ranging spectrum of individuals presenting with RCRD. Clinicians should use their communication and rehabilitation expertise to plan an exercise-based program in conjunction with the individual with RCRSD, which is regularly reviewed and adjusted.

Reconstitution and optimisation of the biosynthesis of bacterial sugar pseudaminic acid (Pse5Ac7Ac) enables preparative enzymatic synthesis of CMP-Pse5Ac7Ac.

Pseudaminic acids present on the surface of pathogenic bacteria, including gut pathogens Campylobacter jejuni and Helicobacter pylori, are postulated to play influential roles in the etiology of associated infectious diseases through modulating flagella assembly and recognition of bacteria by the human immune system. Yet they are underexplored compared to other areas of glycoscience, in particular enzymes responsible for the glycosyltransfer of these sugars in bacteria are still to be unambiguously characterised. This can be largely attributed to a lack of access to nucleotide-activated pseudaminic acid glycosyl donors, such as CMP-Pse5Ac7Ac. Herein we reconstitute the biosynthesis of Pse5Ac7Ac in vitro using enzymes from C. jejuni (PseBCHGI) in the process optimising coupled turnover with PseBC using deuterium wash in experiments, and establishing a method for co-factor regeneration in PseH tunover. Furthermore we establish conditions for purification of a soluble CMP-Pse5Ac7Ac synthetase enzyme PseF from Aeromonas caviae and utilise it in combination with the C. jejuni enzymes to achieve practical preparative synthesis of CMP-Pse5Ac7Ac in vitro, facilitating future biological studies.

Using automated glycan assembly (AGA) for the practical synthesis of heparan sulfate oligosaccharide precursors.

Herein we report synthesis of complex heparan sulfate oligosaccharide precursors by automated glycan assembly using disaccharide donor building blocks. Rapid access to a hexasaccharide was achieved through iterative solid phase glycosylations on a photolabile resin using Glyconeer™, an automated oligosaccharide synthesiser, followed by photochemical cleavage and glycan purification using simple flash column chromatography.

Site-selective C-C modification of proteins at neutral pH using organocatalyst-mediated cross aldol ligations.

The bioconjugation of proteins with small molecules has proved an invaluable strategy for probing and perturbing biological mechanisms. The general use of chemical methods for protein functionalisation can be limited however by the requirement for complicated reaction partners to be present in large excess, and harsh conditions which are incompatible with many protein scaffolds. Herein we describe a site-selective organocatalyst-mediated protein aldol ligation (OPAL) that affords stable carbon-carbon linked bioconjugates at neutral pH. OPAL enables rapid modification of proteins using simple aldehyde probes in minimal excess, and is utilised here in the affinity tagging of proteins in cell lysate. Furthermore we demonstrate that the ß-hydroxy aldehyde OPAL product can be functionalised again at neutral pH in a tandem organocatalyst-mediated oxime ligation. This tandem strategy is showcased in the 'chemical mimicry' of a previously inaccessible natural dual post-translationally modified protein integral to the pathogenesis of the neglected tropical disease Leishmaniasis.

Conserved Histidine Adjacent to the Proximal Cluster Tunes the Anaerobic Reductive Activation of Escherichia coli Membrane-Bound [NiFe] Hydrogenase-1.

[NiFe] hydrogenases are electrocatalysts that oxidize H2 at a rapid rate without the need for precious metals. All membrane-bound [NiFe] hydrogenases (MBH) possess a histidine residue that points to the electron-transfer iron sulfur cluster closest ("proximal") to the [NiFe] H2-binding active site. Replacement of this amino acid with alanine induces O2 sensitivity, and this has been attributed to the role of the histidine in enabling the reversible O2-induced over-oxidation of the [Fe4S3Cys2] proximal cluster possessed by all O2-tolerant MBH. We have created an Escherichia coli Hyd-1 His-to-Ala variant and report O2-free electrochemical measurements at high potential that indicate the histidine-mediated [Fe4S3Cys2] cluster-opening/closing mechanism also underpins anaerobic reactivation. We validate these experiments by comparing them to the impact of an analogous His-to-Ala replacement in Escherichia coli Hyd-2, a [NiFe]-MBH that contains a [Fe4S4] center.

Palladium-unleashed proteins: gentle aldehyde decaging for site-selective protein modification.

Protein bioconjugation frequently makes use of aldehydes as reactive handles, with methods for their installation being highly valued. Here a new, powerful strategy to unmask a reactive protein aldehyde is presented. A genetically encoded caged glyoxyl aldehyde, situated in solvent-accessible locations, can be rapidly decaged through treatment with just one equivalent of allylpalladium(ii) chloride dimer at physiological pH. The protein aldehyde can undergo subsequent oxime ligation for site-selective protein modification. Quick yet mild conditions, orthogonality and powerful exposed reactivity make this strategy of great potential in protein modification.

Retuning the Catalytic Bias and Overpotential of a [NiFe]-Hydrogenase via a Single Amino Acid Exchange at the Electron Entry/Exit Site.

The redox chemistry of the electron entry/exit site in Escherichia coli hydrogenase-1 is shown to play a vital role in tuning biocatalysis. Inspired by nature, we generate a HyaA-R193L variant to disrupt a proposed Arg-His cation-π interaction in the secondary coordination sphere of the outermost, "distal", iron-sulfur cluster. This rewires the enzyme, enhancing the relative rate of H2 production and the thermodynamic efficiency of H2 oxidation catalysis. On the basis of Fourier transformed alternating current voltammetry measurements, we relate these changes in catalysis to a shift in the distal [Fe4S4]2+/1+ redox potential, a previously experimentally inaccessible parameter. Thus, metalloenzyme chemistry is shown to be tuned by the second coordination sphere of an electron transfer site distant from the catalytic center.

Biosynthesis of Salmonella enterica [NiFe]-hydrogenase-5: probing the roles of system-specific accessory proteins.

A subset of bacterial [NiFe]-hydrogenases have been shown to be capable of activating dihydrogen-catalysis under aerobic conditions; however, it remains relatively unclear how the assembly and activation of these enzymes is carried out in the presence of air. Acquiring this knowledge is important if a generic method for achieving production of O2-resistant [NiFe]-hydrogenases within heterologous hosts is to be developed. Salmonella enterica serovar Typhimurium synthesizes the [NiFe]-hydrogenase-5 (Hyd-5) enzyme under aerobic conditions. As well as structural genes, the Hyd-5 operon also contains several accessory genes that are predicted to be involved in different stages of biosynthesis of the enzyme. In this work, deletions in the hydF, hydG, and hydH genes have been constructed. The hydF gene encodes a protein related to Ralstonia eutropha HoxO, which is known to interact with the small subunit of a [NiFe]-hydrogenase. HydG is predicted to be a fusion of the R. eutropha HoxQ and HoxR proteins, both of which have been implicated in the biosynthesis of an O2-tolerant hydrogenase, and HydH is a homologue of R. eutropha HoxV, which is a scaffold for [NiFe] cofactor assembly. It is shown here that HydG and HydH play essential roles in Hyd-5 biosynthesis. Hyd-5 can be isolated and characterized from a ΔhydF strain, indicating that HydF may not play the same vital role as the orthologous HoxO. This study, therefore, emphasises differences that can be observed when comparing the function of hydrogenase maturases in different biological systems.

Physiotherapy assessment of shoulder stiffness and how it influences management.

Common causes of shoulder stiffness include osteoarthritis, trauma, rheumatological conditions and stiffness secondary to soft tissue adaptation. Physiotherapy assessment of the stiff shoulder aims to ascertain the key causative factors of stiffness to inform effective management planning. Identification of whether a patient presents with pain or stiffness as their predominant symptom further guides treatment selection. The current evidence base underpins a management algorithm which has been developed to guide the assessment and management of patients presenting with shoulder stiffness.

Autophagy is a cell death mechanism in Toxoplasma gondii.

Nutrient sensing and the capacity to respond to starvation is tightly regulated as a means of cell survival. Among the features of the starvation response are induction of both translational repression and autophagy. Despite the fact that intracellular parasite like Toxoplasma gondii within a host cell predicted to be nutrient rich, they encode genes involved in both translational repression and autophagy. We therefore examined the consequence of starvation, a classic trigger of autophagy, on intracellular parasites. As expected, starvation results in the activation of the translational repression system as evidenced by elevation of phosphorylated TgIF2α (TgIF2α-P). Surprisingly, we also observe a rapid and selective fragmentation of the single parasite mitochondrion that leads irreversibly to parasite death. This profound effect was dependent primarily on the limitation of amino acids and involved signalling by the parasite TOR homologue. Notably, the effective blockade of mitochondrial fragmentation by the autophagy inhibitor 3-methyl adenine (3-MA) suggests an autophagic mechanism. In the absence of a documented apoptotic cascade in T. gondii, the data suggest that autophagy is the primary mechanism of programmed cell death in T. gondii and potentially other related parasites.

Expression of protein complexes using multiple Escherichia coli protein co-expression systems: a benchmarking study.

Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes.

Structure and kinetic investigation of Streptococcus pyogenes family GH38 alpha-mannosidase.

BACKGROUND: The enzymatic hydrolysis of alpha-mannosides is catalyzed by glycoside hydrolases (GH), termed alpha-mannosidases. These enzymes are found in different GH sequence-based families. Considerable research has probed the role of higher eukaryotic "GH38" alpha-mannosides that play a key role in the modification and diversification of hybrid N-glycans; processes with strong cellular links to cancer and autoimmune disease. The most extensively studied of these enzymes is the Drosophila GH38 alpha-mannosidase II, which has been shown to be a retaining alpha-mannosidase that targets both alpha-1,3 and alpha-1,6 mannosyl linkages, an activity that enables the enzyme to process GlcNAc(Man)(5)(GlcNAc)(2) hybrid N-glycans to GlcNAc(Man)(3)(GlcNAc)(2). Far less well understood is the observation that many bacterial species, predominantly but not exclusively pathogens and symbionts, also possess putative GH38 alpha-mannosidases whose activity and specificity is unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that the Streptococcus pyogenes (M1 GAS SF370) GH38 enzyme (Spy1604; hereafter SpGH38) is an alpha-mannosidase with specificity for alpha-1,3 mannosidic linkages. The 3D X-ray structure of SpGH38, obtained in native form at 1.9 A resolution and in complex with the inhibitor swainsonine (K(i) 18 microM) at 2.6 A, reveals a canonical GH38 five-domain structure in which the catalytic "-1" subsite shows high similarity with the Drosophila enzyme, including the catalytic Zn(2+) ion. In contrast, the "leaving group" subsites of SpGH38 display considerable differences to the higher eukaryotic GH38s; features that contribute to their apparent specificity. CONCLUSIONS/SIGNIFICANCE: Although the in vivo function of this streptococcal GH38 alpha-mannosidase remains unknown, it is shown to be an alpha-mannosidase active on N-glycans. SpGH38 lies on an operon that also contains the GH84 hexosaminidase (Spy1600) and an additional putative glycosidase. The activity of SpGH38, together with its genomic context, strongly hints at a function in the degradation of host N- or possibly O-glycans. The absence of any classical signal peptide further suggests that SpGH38 may be intracellular, perhaps functioning in the subsequent degradation of extracellular host glycans following their initial digestion by secreted glycosidases.

A comparison of methods for shoulder strength assessment and analysis of Constant score change in patients aged over fifty years in the United Kingdom.

Normal values for shoulder assessment are imperative for the diagnosis of pathology and measurement of treatment outcome. Normal values for the United Kingdom are currently not known. This study comprised 108 control patients aged over 50 years who were under the care of one general practitioner, and the Constant score was measured. The Constant score was assessed via 3 techniques for strength measurement: maximum strength with myometer, mean strength with myometer, and maximum strength with fixed spring balance. Myometer maximum strength values were very similar to fixed spring balance maximum strength values, with a mean difference of 0.5. Myometer mean strength was lower than myometer maximum strength by 3 points. Age and sex both significantly affected Constant score (P < .001 for both). The Constant score fell by 0.3 points per year over 50 years of age. Men had a score that was 7.5 points greater than that in women. The Constant score decreases predictably with age in the United Kingdom. Methods of strength assessment are not the same. A uniform method of shoulder strength assessment is required to allow for meaningful comparisons between series.