Pockets as structural descriptors of EGFR kinase conformations

Epidermal Growth Factor Receptor (EGFR), a tyrosine kinase receptor, is one of the main tumor markers in different types of cancers. The kinase native state is mainly composed of two populations of conformers: active and inactive. Several sequence variations in EGFR kinase region promote the differential enrichment of conformers with higher activity. Some structural characteristics have been proposed to differentiate kinase conformations, but these considerations could lead to ambiguous classifications. We present a structural characterisation of EGFR kinase conformers, focused on active site pocket comparisons, and the mapping of known pathological sequence variations. A structural based clustering of this pocket accurately discriminates active from inactive, well-characterised conformations. Furthermore, this main pocket contains, or is in close contact with, ≈65% of cancer-related variation positions. Although the relevance of protein dynamics to explain biological function has been extensively recognised, the usage of the ensemble of conformations in dynamic equilibrium to represent the functional state of proteins and the importance of pockets, cavities and/or tunnels was often neglected in previous studies. These functional structures and the equilibrium between them could be structurally analysed in wild type as well as in sequence variants. Our results indicate that biologically important pockets, as well as their shape and dynamics, are central to understanding protein function in wild-type, polymorphic or disease-related variations.Fil: Hasenahuer, Marcia Anahí. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Barletta Roldan, Patricio German. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernández Alberti, Sebastián. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Parisi, Gustavo Daniel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fornasari, Maria Silvina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Crystal structure of an Fe-S cluster-containing fumarate hydratase enzyme from

Fumarate hydratases (FHs) are essential metabolic enzymes grouped into two classes. Here, we present the crystal structure of a class I FH, the cytosolic FH from Leishmania major, which reveals a previously undiscovered protein fold that coordinates a catalytically essential [4Fe-4S] cluster. Our 2.05 Å resolution data further reveal a dimeric architecture for this FH that resembles a heart, with each lobe comprised of two domains that are arranged around the active site. Besides the active site, where the substrate S-malate is bound bidentate to the unique iron of the [4Fe-4S] cluster, other binding pockets are found near the dimeric enzyme interface, some of which are occupied by malonate, shown here to be a weak inhibitor of this enzyme. Taken together, these data provide a framework both for investigations of the class I FH catalytic mechanism and for drug design aimed at fighting neglected tropical diseases

Engineering enzyme access tunnels

Enzymes are efficient and specific catalysts for many essential reactions in biotechnological and pharmaceutical industries. Many times, the natural enzymes do not display the catalytic efficiency, stability or specificity required for these industrial processes. The current enzyme engineering methods offer solutions to this problem, but they mainly target the buried active site where the chemical reaction takes place. Despite being many times ignored, the tunnels and channels connecting the environment with the active site are equally important for the catalytic properties of enzymes. Changes in the enzymatic tunnels and channels affect enzyme activity, specificity, promiscuity, enantioselectivity and stability. This review provides an overview of the emerging field of enzyme access tunnel engineering with case studies describing design of all the aforementioned properties. The software tools for the analysis of geometry and function of the enzymatic tunnels and channels and for the rational design of tunnel modifications will also be discussed. The combination of new software tools and enzyme engineering strategies will provide enzymes with access tunnels and channels specifically tailored for individual industrial processes

Valorization of Monolignols through Enzymatic Bioconversions by Rational Design of Cytochrome P450 BM3

Lignin valorization is crucial to replace fossil fuels for a more sustainable future (Karunarathna & Smith, 2020). Reductive catalysis fractionation (RCF) produces phenolic monomers, termed monolignols, that can be valorized through enzymatic bioconversions. This study’s main aim was to increase the value of three RCF monolignols: 4-propyl phenol (4-PP), 4-propyl guaiacol (4-PG), and 4-propyl syringol (4-PS), through oxyfunctionalization to value-added aromatic chemicals employing cytochrome P450 BM3 (CYPBM3) of Bacillus megaterium. Cytochrome P450 enzymes (CYPs) play critical roles in a wide range of biological processes by catalyzing diverse reactions (Munro et al., 2002) and we sought to modulate the substrate specificity and selectivity of CYPBM3 by rational design. Three mutants, M1 (Ala184Phe), M5 (Ala74Gly), and M7 (Ala328Leu), and CYPBM3wt were expressed and purified based on initial activity towards the monolignols and expression yields. Binding to monolignols was evaluated using absorbance shift assays, activity on monolignols was assessed via NADPH depletion assay, and oxidative regioselectivity was determined through mass spectrometry (MS) analysis. Binding shift assays indicated that M5 had improved binding to the bulkier monolignols (4-PG and 4-PS), while for M1 binding was only observed with the native palmitic acid substrate. CYPBM3wt binding was observed with all substrates, while M7 did not show any binding with any substrate potentially due to the introduction of steric hindrance in the active site. M5 showed the highest NADPH consumption rate with all three monolignols. M1 showed the lowest NADPH consumption rates with 4-PP and 4-PS. M7 showed the highest NADPH consumption rate with palmitic acid, exceeding the wild type, which could be the result of the uncoupling of electron donation from the reductase domain. 4-PP was the only monolignol showing product formation by identification by MS-analysis displaying hydroxylation in the meta- and ortho positions of the benzene ring. 4-PG also showed hydroxylation but in an unknown position. 4-PS had no product formation indicating uncoupling of electrons and non-productive binding. The results of these analyses support the use of CYPBM3 for the oxyfunctionalization of lignin-derived monolignols. Further studies will be necessary to correlate substrate binding activity or uncoupling to better conclude which candidate had better activity. LC-MS should be employed for quantification of substrate disappearance and product formation to evaluate activity and specificity. Informed by this, a combination of mutations could also be generated for improved activity

Small surface, big effects, and big challenges: toward understanding enzymatic activity at the inorganic nanoparticle–substrate interface

Enzymes are important biomarkers for molecular diagnostics and targets for the action of drugs. In turn, inorganic nanoparticles (NPs) are of interest as materials for biological assays, biosensors, cellular and in vivo imaging probes, and vectors for drug delivery and theranostics. So how does an enzyme interact with a NP, and what are the outcomes of multivalent conjugation of its substrate to a NP? This invited feature article addresses the current state of the art in answering this question. Using gold nanoparticles (Au NPs) and semiconductor quantum dots (QDs) as illustrative materials, we discuss aspects of enzyme structure–function and the properties of NP interfaces and surface chemistry that determine enzyme–NP interactions. These aspects render the substrate-on-NP configurations far more complex and heterogeneous than the conventional turnover of discrete substrate molecules in bulk solution. Special attention is also given to the limitations of a standard kinetic analysis of the enzymatic turnover of these configurations, the need for a well-defined model of turnover, and whether a “hopping” model can account for behaviors such as the apparent acceleration of enzyme activity. A detailed and predictive understanding of how enzymes turn over multivalent NP-substrate conjugates will require a convergence of many concepts and tools from biochemistry, materials, and interface science. In turn, this understanding will help to enable rational, optimized, and value-added designs of NP bioconjugates for biomedical and clinical applications

Coupling Dynamics and Evolutionary Information with Structure to Identify Protein Regulatory and Functional Binding Sites

Binding sites in proteins can be either specifically functional binding sites (active sites) that bind specific substrates with high affinity or regulatory binding sites (allosteric sites), that modulate the activity of functional binding sites through effector molecules. Owing to their significance in determining protein function, the identification of protein functional and regulatory binding sites is widely acknowledged as an important biological problem. In this work, we present a novel binding site prediction method, AR-Pred (Active and Regulatory site Prediction), which supplements protein geometry, evolutionary and physicochemical features with information about protein dynamics to predict putative active and allosteric site residues. Since the intrinsic dynamics of globular proteins plays an essential role in controlling binding events, we find it to be an important feature for the identification of protein binding sites. We train and validate our predictive models on multiple balanced training and validation sets with random forest machine learning and obtain an ensemble of discrete models for each prediction type. Our models for active site prediction yield a median AUC of 91% and MCC of 0.68, whereas the less welldefined allosteric sites are predicted at a lower level with a median AUC of 80% and MCC of 0.48. When tested on an independent set of proteins, our models for active site prediction show comparable performance to two existing methods and gains compared to two others, while the allosteric site models show gains when tested against three existing prediction methods. AR-Pred is available as a free downloadable package at https://github.com/sambitmishra0628/ARPRED_ source

Isolation, overexpression and characterization of an alkaline˗stable lipase KV1 from acinetobacter haemolyticus

The study reports the purification and comprehensive biochemical characterization of a novel lipase KV1 (LipKV1) from Acinetobacter haemolyticus strain KV1. Strain KV1 was identified as Acinetobacter haemolyticus based on results of 16S rDNA sequencing, phylogenetic and BIOLOG. The intracellular wild-type LipKV1 was purified to offer specific activity of 32 U/mg with an estimated relative molecular mass of 37 kDa. The PCR product of LipKV1 revealed that the retrieved sequence contained the proposed complete lipase gene sequence at nucleic acid positions 1~954. The purified wild-type LipKV1 exhibited a maximum relative activity at 40°C and pH 8.0. The lipase was activated (112-128%) in Na+, Ca2+, K+ and Mg2+ and the enzyme hydrolyzed a wide range of oils with tributyrin (140%) being the preferred ones. Reducing (PMSF, DTT, β-mercaptoethanol) and chelating (EDTA) agents significantly inhibited the LipKV1 relative activity (p < 0.05). Surfactants Tween 20-80 (110-143%) significantly enhanced the relative activity (p < 0.05). Gene encoding intracellular lipase was cloned to produce a large quantity of the recombinant LipKV1. The lipase which contained His-tag was expressed in Esherichia coli BL21 (DE3) cells using pET-30a as expression vector. Using the central composite design, screening and optimization of induction conditions (cell density before induction, IPTG concentration, post-induction temperature and post-induction time) were performed. All parameters were significant (p < 0.05) in influencing the expression of LipKV1, rendering a 70% increase in enzyme production at optimum induction conditions. The expressed recombinant LipKV1 was purified using Ni-affinity chromatography, to a specific activity of 233.4 U/mg and an estimated relative molecular mass of 39 kDa. The recombinant LipKV1 exhibited a maximum activity at 40°C and pH 8.0. Homology modeling of the lipase structure was carried out based on the template structure of a carboxylesterase from the archaeon Archaeoglobus fulgidus, which shares a 58% sequence identity to LipKV1. The LipKV1 model comprised a single compact domain consisting of seven parallel and one anti-parallel β-strand surrounded by nine α-helices. Three conserved active-site residues, namely Ser165, Asp259, and His289, and a tunnel through which substrates access the binding site were identified. Docking of the substrates tributyrin and palmitic acid into the active site of LipKV1 modeled at pH 8.0 revealed an aromatic platform responsible for the substrate recognition and preference towards tributyrin. The binding modes from the docking simulation appear to correlate well with the experimentally determined hydrolysis pattern, for which pH 8.0 is optimum and tributyrin being the preferred substrate. A low Km value (0.6 mM) for tributyrin further verifies the high affinity of LipKV1 for the substrate. Biophysical characterization of recombinant LipKV1 protein using ultaviolet-visible (UV-Vis) spectroscopy, circular dichroism (CD), fluorescence spectroscopy, ANS fluorescence spectroscopy, Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) indicated that the lipase retains its secondary structure and good folding at alkaline pH conditions (pH 8.0 and pH 12.0) and at 40C. Alkaline-stable enzymes such as LipKV1 are therefore, useful in biotechnology-based industries in order to shorten production time, minimizing energy consumption and preventing undesired chemical transformations

Development of computational tools for modeling the biotransport of small organic molecules into the active site of broad-substrate specificity enzymes

University of Minnesota Ph.D. dissertation. July 2019. Major: Mechanical Engineering. Advisors: Alptekin Aksan, Lawrence Wackett. 1 computer file (PDF); xii, 248 pages.In this dissertation research, two new computational tools were developed to model the biotransport of small organic molecules into the active site of broad-substrate specificity (BSS) enzymes. The biological organism selected to develop, test and validate these tools were Rieske non-heme iron dioxygenases. Members of this family of enzymes are known to have biocatalytic activity on more than three hundred different substrates. The large diversity of substrates that can be acted upon makes these enzymes very attractive in biotechnological processes such as bioremediation. In addition, the highly specific chirality of the products obtained makes these enzymes attractive for the potential synthesis of pharmaceutical precursors. Currently, the most common way to identify new substrates requires formulating an educated guess followed by the arduous task of testing each possible compound individually. This slows down the pace at which new industrial processes can be formulated or current ones further developed. The tools presented in this research provide fundamental and practical scientific contributions. For the basic science studies of my dissertation, an all-atom and, a coarse-grained (CG) model of Rieske non-heme iron dioxygenases were used to investigate the factors that affect the biotransport of small organic molecules into their active sites. From the all-atom model I discovered a gating mechanism that allows aromatic substrates into the active site and blocks other compounds. The key to these gates are T-stacked pi-pi interactions between hydrophobic amino acids and the aromatic substrates. On the other hand, from the CG model I discovered that the shape of tunnel modulates the hydrophobicity level of the surface. As the tunnels become more concave, the hydrophobicity increases causing the formation of a water exclusion zone which increases the diffusivity of aromatic substrates. The CG models also revealed that convex tunnels prevent the adhesion of hydrophobic substrates to the tunnel walls; providing a possible explanation for the evolution of bottlenecks at the entrance of Rieske active sites. For the practical contributions of my dissertation, I developed two new computational tools for the prediction of Rieske substrates. The first tool is an all-atom algorithm that models the stochastic roto-translational movement of small organic molecules along the Rieske enzyme tunnels. This algorithm has a 92% prediction accuracy of Rieske substrates. In addition, it is capable of elucidating the location of high-energy barriers along the tunnel, allowing the formulation of possible protein engineering sites. The second tool is a CG non dimensional model of the Rieske enzyme tunnels. This algorithm has a 90% prediction accuracy of Rieske substrates. The processing time of 1ms/substrate combined with its high accuracy allows for the high-throughput screening of possible Rieske substrates

Electrocatalytic cascade reactions by nanoconfinement of intermediates: the case of electrochemical CO2 reduction

Integral to the activity of enzymes is their architecture: an active site located at the base of an isolated substrate channel.1,2 Enzymes are also able to produce highly complex molecules with high efficiency because of their ability to perform cascade reactions via substrate channelling.3 Using substrate channelling, two different active sites can be connected via a tunnel, or substrate channel, that allows the diffusion of intermediates to the next active site without diffusion to the bulk solution.4,5 The imitation of enzyme architecture was previously applied to nanoparticle electrocatalysts called nanozymes by locating Pt active sites inside isolated substrate channels of PtNi nanoparticles.6 The active sites inside the channels were more active than those on the exterior.6 The enhanced activity was attributed to a nanoconfinement effect, the overlap of electrical double layers enhancing the flux of reactant protons by migration.7 The next step is the development of nanozymes for two-step cascade reactions. Electrochemical CO2 reduction is a suitable model reaction for cascade reactions, given it is well known that the reaction can be divided into two steps with CO as the key intermediate.8 It is also well known that metals like Ag can convert CO2 to CO at low overpotentials9 and Cu can convert CO to more complex organic molecules at low overpotentials10 but not vice versa. We designed an electrocatalytic nanoparticle comprising an accessible Ag core and a porous Cu shell, called a cascade nanozyme.11 Ag acts as the first active site where CO2 is reduced to CO. Cu active sites on the porous shell function as the second site where CO can be further reduced to more complex organic molecules, like ethylene, ethanol and propanol. In this work, cascade nanozymes were synthesised and characterised for electrochemical CO2 reduction using GC-FID and 1H-NMR. C3H8O was formed at -0.6 V vs. RHE on the cascade nanozymes, where an equivalent Cu-only control showed no activity for C3 products.11 A combination of in-situ (applied for the first time to CO2 reduction) and ex-situ TEM were used to characterise the AgCu nanozymes in electrochemical CO2 reduction conditions.12 It was found that a combination of applied potential and (local) CO concentration had the greatest impact upon stability. Using this knowledge, PdCu nanozymes were also synthesised as a comparison and showed improved stability at -0.8 V vs RHE compared to AgCu nanozymes, which was attributed to the lower CO production rate of Pd compared to Ag. Cascade nanozymes with different Cu shell thicknesses were synthesised and characterised for electrochemical CO¬2 reduction.13 Thin shells were more active for C2+ products at -0.60 V vs RHE but thick shells were more active at -0.65 and -0.70 V vs RHE. These results illustrate the importance of nanoconfinement for controlling the intermediate in a two-step reaction, in this case CO in CO2 reduction. Electrocatalytic nanoparticle architecture can be used to confine intermediates from one active site to another. In electrochemical CO2 reduction, such substrate channelling can create high local concentrations of important intermediates like CO. We have found that such high local can enhance the selectivity for longer chain hydrocarbons and also influence structural stability. Control can be exerted over the intermediate (CO) and the resultant products by changing the length of the substrate channel. These results illustrate the influence that intermediates supplied in cascade reactions can have upon activity, selectivity and stability. References (1) Kingsley, L. J.; Lill, M. A. Substrate Tunnels in Enzymes: Structure-Function Relationships and Computational Methodology. Proteins Struct. Funct. Bioinforma. 2015, 83 (4), 599–611. (2) Prokop, Z.; Gora, A.; Brezovsky, J.; Chaloupkova, R.; Stepankova, V.; Damborsky, J. Engineering of Protein Tunnels : The Keyhole – Lock – Key Model for Catalysis by Enzymes with Buried Active Sites. In Protein engineering handbook; Lutz, S., Theo Bornscheuer, U., Eds.; Wiley-VCH, Weinheim, 2012; Vol. 3, pp 421–464. (3) Wheeldon, I.; Minteer, S. D.; Banta, S.; Barton, S. C.; Atanassov, P.; Sigman, M. Substrate Channelling as an Approach to Cascade Reactions. Nature Chemistry. Nature Publishing Group March 22, 2016, pp 299–309. (4) Spivey, H. O.; Ovádi, J. Substrate Channeling. Methods: A Companion to Methods in Enzymology. Academic Press October 1, 1999, pp 306–321. (5) Miles, E. W.; Rhee, S.; Davies, D. R. The Molecular Basis of Substrate Channeling. Journal of Biological Chemistry. American Society for Biochemistry and Molecular Biology April 30, 1999, pp 12193–12196. (6) Benedetti, T. M.; Andronescu, C.; Cheong, S.; Wilde, P.; Wordsworth, J.; Kientz, M.; Tilley, R. D.; Schuhmann, W.; Gooding, J. J. Electrocatalytic Nanoparticles That Mimic the Three-Dimensional Geometric Architecture of Enzymes: Nanozymes. J. Am. Chem. Soc. 2018, 140 (41), 13449–13455. (7) Wordsworth, J.; Benedetti, T. M.; Alinezhad, A.; Tilley, R. D.; Edwards, M. A.; Schuhmann, W.; Gooding, J. J. The Importance of Nanoscale Confinement to Electrocatalytic Performance. Chem. Sci. 2020. (8) Jouny, M.; Hutchings, G. S.; Jiao, F. Carbon Monoxide Electroreduction as an Emerging Platform for Carbon Utilization. Nature Catalysis. Nature Research December 1, 2019, pp 1062–1070. (9) Lu, Q.; Rosen, J.; Zhou, Y.; Hutchings, G. S.; Kimmel, Y. C.; Chen, J. G.; Jiao, F. A Selective and Efficient Electrocatalyst for Carbon Dioxide Reduction. Nat. Commun. 2014, 5, 3242. (10) Li, C. W.; Ciston, J.; Kanan, M. W. Electroreduction of Carbon Monoxide to Liquid Fuel on Oxide-Derived Nanocrystalline Copper. Nature 2014, 508 (7497), 504–507. (11) O’Mara, P. B.; Wilde, P.; Benedetti, T. M.; Andronescu, C.; Cheong, S.; Gooding, J. J.; Tilley, R. D.; Schuhmann, W. Cascade Reactions in Nanozymes: Spatially Separated Active Sites inside Ag-Core–Porous-Cu-Shell Nanoparticles for Multistep Carbon Dioxide Reduction to Higher Organic Molecules. J. Am. Chem. Soc. 2019, 141 (36), 14093–14097. (12) Wilde, P.; O’Mara, P. B.; Junqueira, J. R. C.; Tarnev, T.; Benedetti, T. M.; Andronescu, C.; Chen, Y. T.; Tilley, R. D.; Schuhmann, W.; Gooding, J. J. Is Cu Instability during the CO2 reduction Reaction Governed by the Applied Potential or the Local CO Concentration? Chem. Sci. 2021, 12 (11), 4028–4033. (13) O’Mara, P. B.; Somerville, S.; Benedetti, T. M.; Cheong, S.; Chen, H.-S.; Wilde, P.; Schuhmann, W.; Tilley, R.; Gooding, J. Understanding the Influence of Substrate Channel Length on Cascade Reactions for Electrochemical CO2 Reduction. Manuscript in preparatio

Structure-based Enzyme Engineering of Glycosyltransferases

Background: Plant UDP-dependent glycosyltransferases (UGTs) play important roles in biology via the glycosylation of secondary metabolites. The future prospects of UGTs look promising, for example, it may serve as promising path for progress in expanding drug targets and synthesising glycan-based drug with enhanced bioactivity. Nevertheless, the current poor understanding of UGTs at molecular level (e.g. kinetic and structure) has led to a limited understanding of their biological roles and has also hampered their potential applications. Aims: This project aims to 1) build up a mass spectrometry (MS) based approach to study families of UGTs including their substrate specificities, kinetic parameters and mechanisms of action (Chapter 3); 2) identify catalytic key amino acids (ckAAs) in the various UGTs (Chapter 4); and finally, 3) apply the methods above in the study of selected Rhamnosyltransferases (RhaTs) 78D1 and 89C1 (Chapter 5). Methodology: A triple quadrupole MS (QQQ-MS) was used as this instrument required limited modification of substrates and provided direct monitoring of the glycosylated product. ‘Full scan mode’ gave the initial screening of any potential glycosylated product, and the ‘product ion’ mode provided additional confirmation of the formation of the glycosylated product. The ‘multiple reaction monitoring (MRM)’ mode quantified the products formation as a function of reaction time and provided kinetic data of the UGTs (Chapter 3). The study of catalytic key amino acids (ckAAs) was based on the multiple sequence alignment (MSA) method via the AA sequence comparison with template UGTs that have known crystal structures. Subsequent site-directed mutagenesis (SDM) was used to substantiate/disprove the functional role of potential ckAAs: mutants (with potential ckAAs mutated) were checked by MS to find out whether the original activities were maintained and/or new activities were gained. A further activity comparison (kcat/KM) between the active mutant and the wild type (WT) could indicate the influence from a particular AA (Chapter 4). Results and conclusions: 29 recombinant UGTs from groups B, D, F, H and L were examined. New donor activities (e.g. UDP-GlcNAc towards 73B4 and 78D2) were reported. Full kinetic studies of these WT UGTs indicated that they followed the Bi-Bi sequential mechanism. Whilst most followed the Bi-Bi random sequential mechanism, exceptions could be found such as that for 73B4 facilitating the UDP-GlcNAc reaction (Chapter 3). Based on the interactions between the donor and template UGT (e.g. VvGT1, PDB 2C1Z), mutations of the potential ckAAs oriented towards the sugar (positions C2, C3, C4 and C6), phosphate and uridine were designed with alanine and an AA with a similar structural and chemical character. An activity comparison (kcat/KM) between the WT and active mutants indicated that most of the potential ckAAs within the PSPG motif inferred from MSA, were conserved and possibly followed a similar interaction pattern. However, exceptions could be found (e.g. 78D2 D380). Taking the results of both MSA and SDM together, the ckAAs in the active sites in each target UGT towards a specific donor were identified (Chapters 4 and 5). Additionally, the study of Rhamnosyltransferases (RhaT) 78D1 and 89C1 showed that new activities were acquired by point mutagenesis: 78D1 N375Q acquired UDP-Glc and UDP-GlcNAc activities; and 89C1 H357Q acquired UDP-Glc activity (Chapter 5)