Trapping and Characterization of the Reaction Intermediate in Cyclodextrin Glycosyltransferase by Use of Activated Substrates and a Mutant Enzyme

Cyclodextrin glycosyltransferases (CGTases) catalyze the degradation of starch into linear or cyclic oligosaccharides via a glycosyl transfer reaction occurring with retention of anomeric configuration. They are also shown to catalyze the coupling of maltooligosaccharyl fluorides. Reaction is thought to proceed via a double-displacement mechanism involving a covalent glycosyl-enzyme intermediate. This intermediate can be trapped by use of 4-deoxymaltotriosyl α-fluoride (4DG3αF). This substrate contains a good leaving group, fluoride, thus facilitating formation of the intermediate, but cannot undergo the transglycosylation step since the nucleophilic hydroxyl group at the 4-position is missing. When 4DG3αF was reacted with wild-type CGTase (Bacillus circulans 251), it was found to be a slow substrate (kcat = 2 s-1) compared with the parent glycosyl fluoride, maltotriosyl R-fluoride (kcat = 275 s-1). Unfortunately, a competing hydrolysis reaction reduces the lifetime of the intermediate precluding its trapping and identification. However, when 4DG3αF was used in the presence of the presumed acid/base catalyst mutant Glu257Gln, the intermediate could be trapped and analyzed because the first step remained fast while the second step was further slowed (kcat = 0.6 s-1). Two glycosylated peptides were identified in a proteolytic digest of the inhibited enzyme by means of neutral loss tandem mass spectrometry. Edman sequencing of these labeled peptides allowed identification of Asp229 as the catalytic nucleophile and provided evidence for a covalent intermediate in CGTase. Asp229 is found to be conserved in all members of the family 13 glycosyl transferases.

Functional metagenomic screening approach for discovery of new glycoside phosphorylases

Glycoside phosphorylases (GPs) have recently been recognized as potentially useful biocatalysts for the synthesis and biotransformation of glycans. These enzymes ordinarily carry out phosphorolysis of the glycosidic linkage by transferring a glycosyl moiety from the non-reducing end of a di- or polysaccharide substrate onto inorganic phosphate, thereby cleaving the glycosidic bond and generating a sugar-1-phosphate. GPs distinguish themselves from most carbohydrate-active enzymes in that the hydrolytic free energy associated with the ester-linkage of the sugar-1-phosphate product is roughly equivalent to that of the glycosidic linkage in the glycan substrate. Therefore, the equilibrium position can be tipped in favour of glycoside synthesis by manipulation of reaction conditions. GPs thus have considerable potential for the assembly of glycans, especially since their reversibility would allow the use of one GP to degrade an inexpensive glycan to produce a pool of sugar-1-phosphates, while a second GP could be deployed to use those sugar-1-phosphates as donors to synthesize a different, more valuable target glycan. The bottleneck in this approach, however, is the limited range of GPs available, which restricts the classes of glycan that can be assembled. To help increase the spectrum of known GPs available, we have turned to metagenomics as a means to discover new enzymes belonging to this class. We have adapted the molybdenum blue reaction to a high-throughput plate-based metagenomic screen for the discovery of GPs. Our method utilizes the reverse phosphorolysis ability of GPs by coupling inorganic phosphate released during glycan synthesis to the molybdenum blue reaction. Therefore, GP activity can be identified by incubating metagenomic clones with appropriate donor sugar-1-phosphates and acceptor glycans, then monitoring inorganic phosphate accumulation by measuring formation of molybdenum blue. Our pilot screen was optimized to identify cellulose degrading GPs and yielded 7 novel GP ORFs, all from CAZy family GH94. To our knowledge this is the first high-throughput functional metagenomic screen for GP activity. Looking ahead, we have planned to further adapt the screening method so it may identify GP activity from other CAZy families, beyond GH94. The activities that can be identified are dependent on the combination of donor and acceptor substrates used. By mixing and matching different substrates we will be able to narrow or broaden the scope of activities that can be detected within a single screen

Reassessment of Acarbose as a Transition State Analogue Inhibitor of Cyclodextrin Glycosyltransferase

The binding of several different active site mutants of Bacillus circulans cyclodextrin glycosyltransferase to the inhibitor acarbose has been investigated through measurement of Ki values. The mutations represent several key amino acid positions, most of which are believed to play important roles in governing the product specificity of cyclodextrin glycosyltransferase. Michaelis-Menten parameters for the substrates α-maltotriosyl fluoride (αG3F) and α-glucosyl fluoride (αGF) with each mutant have been determined by following the enzyme-catalyzed release of fluoride with an ion-selective fluoride electrode. In both cases, reasonable correlations are observed in logarithmic plots relating the Ki value for acarbose with each mutant and both kcat/Km and Km for the hydrolysis of either substrate by the corresponding mutants. This indicates that acarbose, as an inhibitor, is mimicking aspects of both the ground state and the transition state. A better correlation is observed for αGF (r = 0.98) than αG3F (r = 0.90), which can be explained in terms of the modes of binding of these substrates and acarbose. Re-refinement of the previously determined crystal structure of wild-type CGTase complexed with acarbose reveals a binding mode consistent with the transition state analogue character of this inhibitor.

Tutor-Facilitated Adult Digital Literacy Learning: Insights from a Case Study

While the digital divide has decreased, those who are still unable to access and use information and communication technologies are left further behind. An effective digital literacy program helps underserved populations gain needed skills and alleviate the demand placed on public library staff. This case study presents findings on a digital literacy learning model that utilizes a self-paced online platform and in-person volunteer tutors. The researchers found that the learner/tutor relationship is an essential part of the learning process, and that tutors develop a variety of strategies for helping learners. The researchers also identify aspects of effective program implementation

Towards the on-line development of visual interactive simulation models

Reviews of previous work on visual interactive simulation, and on the interface between humans and computers were undertaken, the latter considering the physical and psychological aspects of the subject. Two simulation projects carried out in association with Rolls-Royce Aero Engines and the British Steel Corporation are described in detail. As a result of these projects and the review of previous studies, a major weakness in the technology of visual interactive simulation was identified: while the visual representation aids validation, verification, and experimentation, no facilities are provided to assist the analyst in the task of model construction. Simulation program generators are of proven use for non-interactive models, but a visual model requires a graphically oriented approach. The main section describes the design and implementation of a substantial extension to the simulation software developed at Warwick. This allows the design and development of displays to be carried out 'on-line', while preserving the one-to-one correspondence between simulation entities and their visual representation. It is suggested that this has the potential to significantly reduce the elapsed time taken to develop visual simulation models, while increasing the involvement of the user (or sponsor) in the modelling process, especially when 'pre-defined' entity types are used to minimise the amount of model- specific coding required. Finally, potential routes for the further development of visual interactive simulation are discussed, including the implementation of a 'simulation language' interpreter within the existing software. This would result in a system which was fully interactive, easing model development as well as experimentation

New enzymes for cell surface modification: Towards universal blood and improved organ transplants

Mammalian cell surfaces are coated in specific sugar structures, many of which function as antigens and are involved in cellular recognition. Important examples are the oligosaccharide A, B, and H antigens present on red blood cells that differentiate the A, B and O blood types. Enzymatic cleavage of the GalNAc and Gal residues from the cell surface would allow conversion of A and B red blood cells, respectively, to O type. Since Type O blood can be universally donated to patients with the same Rh factor, access to efficient enzymes would greatly broaden and simplify blood supply. We have sought such enzymes in metagenomic libraries derived from the human gut microbiome. Please click Additional Files below to see the full abstract

Directed evolution of glycosyltransferase for the artificial biosynthesis of natural product glycosides

Over one fifth natural product drugs (including protein biopharmaceuticals), cosmetics, and nutraceuticals have a diverse set of sugars in their structures. These glycosylations dramatically influence the physicochemical and pharmacological properties of these compounds. Glycosyltransferases (GTs) offer very attractive approaches to the biosynthesis of complex glycosylated natural products. However, the limited number of available GTs, together with their instability and strict substrate specificity, have severely hampered the broad application of these enzymes. In the past few years, we have used directed evolution as a tool to tailor the GTs with desired substrate specificity and higher catalytic activity. Here I will introduce some of our efforts in 1) the semi-rational design of a glucosyltransferase UGT51 from S. cerevisiae to repurposing its promiscuous activity towards the biosynthesis of rare ginsenoside Rh2; and 2) the directed evolution of an α1,3-fucosyltransferase using a single-cell ultrahigh-throughput screening method. I will also discuss the development of new tools for the high-throughput screening method for GTs and the mechanistic insight we found during the evolution of these enzymes

Time, Space, and Monumentality: a few minutes before midnight on Wednesday 4th September 2024

Monumental Process ‘There is Cleopatra’s Needle.’ If we define the Needle in a sufficiently abstract manner we can say that it never changes. But a physicist who looks on that part of the life of nature as a dance of electrons, will tell you that daily it has lost some molecules and gained others, and even the plain man can see that it gets dirtier and is occasionally washed. Thus the question of change in the Needle is a mere matter of definition. The more abstract your definition, the more permanent the Needle. But whether your Needle change or be permanent, all you mean by stating that it is situated on the Charing Cross Embankment, is that amid the structure of events you know of a certain continuous limited stream of events, such that any chunk of that stream, during any hour, or any day, or any second, has the character of being the situation of Cleopatra’s Needle.” (The Concept of Nature: 164) The steady flow of time, is arrested by abstraction and the establishment of cerebral trust to deduce that the needle ‘never changes’. What is perceived in the moment is organized in the mode of presentational immediacy to ensure a degree of order and predictability. In reality, if one stares at Cleopatra’s needle for a protracted period then one is, without being conscious of it, witnessing the degradation or change that exists in the relational exchange between the obelisk and the percipient. This can be confusing and logically at odds with the proposition by Isabelle Stengers that nothing exists outside of visual perception. To be clear, what is meant here is that there does not exist a reality outside vision and another reality inside it. When staring at cleopatra’s needle one may witness a ‘static object’, but equally one is always experiencing an object that is changing, but just at a rate this not immediately perceptible. Either way the same thing, an exchange and processing of data is being enacted and shared. The relationship is a temporal one and as a process it can be embodied and deployed by artists, philosophers or scientists, as advanced beings not given to assumption. Rather than being based on assumption or immediate recognition, their perception is active in a dynamic relational manner that draws as much from the mode of causal efficacy (as temporal and musical/sonic) as it does from that of presentational immediacy (visual). If the process is manifest and experienced consciously then the limiting tendency of symbolic reference can be overcome. Ordinary ‘language’ becomes extraordinary. Rethinking objects/monuments from the perspective of process leads to a Heisenbergian Architecture. Ultimately, considering points as events in a heritage point cloud would mean embracing a view of heritage as an active, open-ended process of meaning-making, rather than a passive, fixed record of bygone times. It would invite a more fluid, reflexive, and creative approach to engaging with the built environment, one that recognises the fundamental entanglement of past and present, material and digital, human and technological. The point cloud becomes not just a model of heritage, but a space of heritage-making in its own right

Captivating the Attention of Strangers: spatial sensory engagement experiments

This presentation demonstrates the interdisciplinary work of the Captivate Heritage Laboratory (University of Greenwich). Operating within a critical framework the work embraces digital aesthetics/ontology and the spatiotemporal weave of ‘permanence’ and transitoriness as it engages with sites and objects of historical significance. Human beings, their ideas, their art, their architecture, their technology, and their social forms of organisation are all systems wherein noise and chaos resolve to some kind of order. These chaotic systems are dynamic, and their spatiotemporal dimensions relative. A critical understanding of this dynamism affords new understandings of our relationship to the material world and to history

Community Connections: Digital Literacy Acquisition Policy Brief

This case study describes how the digital literacy acquisition program in a rural community was sustained through formal and informal connections across a variety of organizations and community institutions. The case study describes the setting, the details of how tutor facilitated, self paced learning was organized, and the various ways connections were made across organizations. Five types of connections are identified: formal top-level partnerships, local connections, library/workforce partnership, school/workforce connections, informal community connections, and lab coordinator/tutor/learner connections. Implications for these findings include how policy makers and other key stakeholders may benefit from recognizing and building on the ways people in the field marshal formal and informal resources to create and sustain vibrant programs