Carbohydrate-binding module tribes.

At present, 69 families of carbohydrate-binding modules (CBMs) have been isolated by statistically significant differences in the amino acid sequences (primary structures) of their members, with most members of different families showing little if any homology. On the other hand, members of the same family have primary and tertiary (three-dimensional) structures that can be computationally aligned, suggesting that they are descended from common protein ancestors. Members of the large majority of CBM families are ß-sandwiches. This raises the question of whether members of different families are descended from distant common ancestors, and therefore are members of the same tribe. We have attacked this problem by attempting to computationally superimpose tertiary structure representatives of each of the 53 CBM families that have members with known tertiary structures. When successful, we have aligned locations of secondary structure elements and determined root mean square deviations and percentages of similarity between adjacent amino acid residues in structures from similar families. Further criteria leading to tribal membership are amino acid chain lengths and bound ligands. These considerations have led us to assign 27 families to nine tribes. Eight of the tribes have members with ß-sandwich structures, while the ninth is composed of structures with ß-trefoils.

Fatty acid synthesis enzyme clans.

Ketoacyl reductases (KRs), hydroxyacyl dehydratases (HDs), and enoyl reductases (ERs) are part of the fatty acid/polyketide synthesis cycle. They are known as acyl dehydrogenases, enoyl hydratases, and hydroxyacyl dehydrogenases, respectively, when catalyzing their reverse reactions. Earlier, we classified these enzymes into four KR, eight HD, and five ER families by statistical criteria. Members of all four KR families and three ER families have Rossmann folds, while five HD family members have HotDog folds. This suggests that those proteins with the same folds in different families may be distantly related, and therefore in clans, even though their amino acid sequences may not be homologous. We have now defined two clans containing three of the four KR families and two of the eight HD families, using manual and statistical tests. One of the ER families is related to the KR clan.

Molecular mechanism of a hotdog-fold acyl-CoA thioesterase.

Thioesterases are enzymes that hydrolyze thioester bonds between a carbonyl group and a sulfur atom. They catalyze key steps in fatty acid biosynthesis and metabolism, as well as polyketide biosynthesis. The reaction molecular mechanism of most hotdog-fold acyl-CoA thioesterases remains unknown, but several hypotheses have been put forward in structural and biochemical investigations. The reaction of a human thioesterase (hTHEM2), representing a thioesterase family with a hotdog fold where a coenzyme A moiety is cleaved, was simulated by quantum mechanics/molecular mechanics metadynamics techniques to elucidate atomic and electronic details of its mechanism, its transition-state conformation, and the free energy landscape of the process. A single-displacement acid-base-like mechanism, in which a nucleophilic water molecule is activated by an aspartate residue acting as a base, was found, confirming previous experimental proposals. The results provide unambiguous evidence of the formation of a tetrahedral-like transition state. They also explain the roles of other conserved active-site residues during the reaction, especially that of a nearby histidine/serine pair that protonates the thioester sulfur atom, the participation of which could not be elucidated from mutation analyses alone.

ThYme: a database for thioester-active enzymes.

The ThYme (Thioester-active enzYme; http://www.enzyme.cbirc.iastate.edu) database has been constructed to bring together amino acid sequences and 3D (tertiary) structures of all the enzymes constituting the fatty acid synthesis and polyketide synthesis cycles. These enzymes are active on thioester-containing substrates, specifically those that are parts of the acyl-CoA synthase, acyl-CoA carboxylase, acyl transferase, ketoacyl synthase, ketoacyl reductase, hydroxyacyl dehydratase, enoyl reductase and thioesterase enzyme groups. These groups have been classified into families, members of which are similar in sequences, tertiary structures and catalytic mechanisms, implying common protein ancestry. ThYme is continually updated as sequences and tertiary structures become available.

COVID-19 vaccine effectiveness and variants in Nepal: study protocol for a test-negative case-control study with SARS-CoV-2 genetic sequencing.

INTRODUCTION: Inactivated, viral vector and mRNA vaccines have been used in the Nepali COVID-19 vaccination programme but there is little evidence on the effectiveness of these vaccines in this setting. The aim of this study is to describe COVID-19 vaccine effectiveness in Nepal and provide information on infections with SARS-CoV-2 variants. METHODS AND ANALYSIS: This is a hospital-based, prospective test-negative case-control study conducted at Patan Hospital, Kathmandu. All patients >18 years of age presenting to Patan Hospital with COVID-19-like symptoms who have received a COVID-19 antigen/PCR test are eligible for inclusion. The primary outcome is vaccine effectiveness of licensed COVID-19 vaccines against laboratory-confirmed COVID-19 disease.After enrolment, information will be collected on vaccine status, date of vaccination, type of vaccine, demographics and other medical comorbidities. The primary outcome of interest is laboratory-confirmed SARS-CoV-2 infection. Cases (positive for SARS-CoV-2) and controls (negative for SARS-CoV-2) will be enrolled in a 1:4 ratio. Vaccine effectiveness against COVID-19 disease will be analysed by comparing vaccination status with SARS-CoV-2 test results.Positive SARS-CoV-2 samples will be sequenced to identify circulating variants and estimate vaccine effectiveness against common variants.Measuring vaccine effectiveness and identifying SARS-CoV-2 variants in Nepal will help to inform public health efforts. Describing disease severity in relation to specific SARS-CoV-2 variants and vaccine status will also inform future prevention and care efforts. ETHICS AND DISSEMINATION: Ethical approval was obtained from the University of Oxford Tropical Ethics Committee (OxTREC) (ref: 561-21) and the Patan Academy of Health Sciences Institutional Review Board (ref: drs2111121578). The protocol and supporting study documents were approved for use by the Nepal Health Research Council (NHRC 550-2021). Results will be disseminated in peer-reviewed journals and to the public health authorities in Nepal.

Structural classification of biotin carboxyl carrier proteins.

We gathered primary and tertiary structures of acyl-CoA carboxylases from public databases, and established that members of their biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains occur in one family each and that members of their carboxyl transferase (CT) domains occur in two families. Protein families have members similar in primary and tertiary structure that probably have descended from the same protein ancestor. The BCCP domains complexed with biotin in acyl and acyl-CoA carboxylases transfer bicarbonate ions from BC domains to CT domains, enabling the latter to carboxylate acyl and acyl-CoA moieties. We separated the BCCP domains into four subfamilies based on more subtle primary structure differences. Members of different BCCP subfamilies often are produced by different types of organisms and are associated with different carboxylases.

Effect of the of 10-valent pneumococcal conjugate vaccine in Nepal 4 years after introduction: an observational cohort study.

BACKGROUND: In Nepal, Streptococcus pneumoniae (pneumococcus) is a common cause of bacterial pneumonia in children, and is a major health concern. There are few data on the effect of vaccination on the disease or colonisation with pneumococci in the nasopharynx of children in this setting. The 10-valent pneumococcal conjugate vaccine (PCV10) was introduced into the routine infant immunisation schedule in Nepal in 2015. We aimed to investigate the effect of the introduction of PCV10 on pneumococcal carriage and disease in children in Nepal. METHODS: We did an observational cohort study in children in Nepal. The hospital surveillance study took place in Patan Hospital, Kathmandu, and community studies in healthy children took place in Kathmandu and Okhaldhunga district. For the surveillance study, all children admitted to Patan Hospital between March 20, 2014, and Dec 31, 2019, aged between 2 months and 14 years with clinician-suspected pneumonia, were eligible for enrolment. For the community study, healthy children aged 0-8 weeks, 6-23 months, and 24-59 months were recruited from Kathmandu, and healthy children aged 6-23 months were recruited from Okhaldhunga. We assessed the programmatic effect of PCV10 introduction using surveillance for nasopharyngeal colonisation, pneumonia, and invasive bacterial disease from 1·5 years before vaccine introduction and 4·5 years after vaccine introduction. For the surveillance study, nasopharyngeal swabs, blood cultures, and chest radiographs were obtained from children admitted to Patan Hospital with suspected pneumonia or invasive bacterial disease. For the community study, nasopharyngeal swabs were obtained from healthy children in the urban and rural settings. Pneumonia outcomes were analysed using log-binomial models and adjusted prevalence ratios (aPR) comparing each calendar year after the introduction of the vaccine into the national programme with the pre-vaccine period (2014-15), adjusted for calendar month, age, and sex. FINDINGS: Between March 20, 2014, and Dec 31, 2019, we enrolled 2051 children with suspected pneumonia, and 11 354 healthy children (8483 children aged 6-23 months, 761 aged 24-59 months, and 2110 aged 0-8 weeks) to assess nasopharyngeal colonisation. Among clinical pneumonia cases younger than 2 years, vaccine serotype carriage declined 82% (aPR 0·18 [95% CI 0·07-0·50]) by 2019. There was no decrease in vaccine serotype carriage in cases among older unvaccinated age groups. Carriage of the additional serotypes in PCV13 was 2·2 times higher by 2019 (aPR 2·17 [95% CI 1·16-4·05]), due to increases in serotypes 19A and 3. Vaccine serotype carriage in healthy children declined by 75% in those aged 6-23 months (aPR 0·25 [95% CI 0·19-0·33]) but not in those aged 24-59 months (aPR 0·59 [0·29-1·19]). A decrease in overall vaccine serotype carriage of 61% by 2019 (aPR 0·39 [95% CI 0·18-0·85]) was also observed in children younger than 8 weeks who were not yet immunised. Carriage of the additional PCV13 serotypes in children aged 6-23 months increased after PCV10 introduction for serotype 3 and 19A, but not for serotype 6A. The proportion of clinical pneumonia cases with endpoint consolidation on chest radiographs declined from 41% in the pre-vaccine period to 25% by 2018, but rose again in 2019 to 36%. INTERPRETATION: The introduction of the PCV10 vaccine into the routine immunisation programme in Nepal has reduced vaccine serotype carriage in both healthy children and children younger than 2 years with pneumonia. Increases in serotypes 19A and 3 highlight the importance of continued surveillance to monitor the effect of vaccine programmes. This analysis demonstrates a robust approach to assessing vaccine effect in situations in which pneumococcal disease endpoint effectiveness studies are not possible. FUNDING: Gavi, the Vaccine Alliance and the World Health Organization.

Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity.

BACKGROUND: Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. RESULTS: To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. CONCLUSION: These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids.

Molecular mechanism of the glycosylation step catalyzed by Golgi alpha-mannosidase II: a QM/MM metadynamics investigation.

Golgi alpha-mannosidase II (GMII), a member of glycoside hydrolase family 38, cleaves two mannosyl residues from GlcNAcMan(5)GlcNAc(2) as part of the N-linked glycosylation pathway. To elucidate the molecular and electronic details of the reaction mechanism, in particular the conformation of the substrate at the transition state, we performed quantum mechanics/molecular mechanics metadynamics simulations of the glycosylation reaction catalyzed by GMII. The calculated free energy of activation for mannosyl glycosylation (23 kcal/mol) agrees very well with experiments, as does the conformation of the glycon mannosyl ring in the product of the glycosylation reaction (the covalent intermediate). In addition, we provide insight into the electronic aspects of the molecular mechanism that were not previously available. We show that the substrate adopts an (O)S(2)/B(2,5) conformation in the GMII Michaelis complex and that the nucleophilic attack occurs before complete departure of the leaving group, consistent with a D(N)A(N) reaction mechanism. The transition state has a clear oxacarbenium ion (OCI) character, with the glycosylation reaction following an (O)S(2)/B(2,5) --> B(2,5) [TS] --> (1)S(5) itinerary, agreeing with an earlier proposal based on comparing alpha- and beta-mannanases. The simulations also demonstrate that an active-site Zn ion helps to lengthen the O2'-H(O2') bond when the substrate acquires OCI character, relieving the electron deficiency of the OCI-like species. Our results can be used to explain the potency of recently formulated GMII anticancer inhibitors, and they are potentially relevant in deriving new inhibitors.

Tertiary structure and characterization of a glycoside hydrolase family 44 endoglucanase from Clostridium acetobutylicum.

A gene encoding a glycoside hydrolase family 44 (GH44) protein from Clostridium acetobutylicum ATCC 824 was synthesized and transformed into Escherichia coli. The previously uncharacterized protein was expressed with a C-terminal His tag and purified by nickel-nitrilotriacetic acid affinity chromatography. Crystallization and X-ray diffraction to a 2.2-A resolution revealed a triose phosphate isomerase (TIM) barrel-like structure with additional Greek key and beta-sandwich folds, similar to other GH44 crystal structures. The enzyme hydrolyzes cellotetraose and larger cellooligosaccharides, yielding an unbalanced product distribution, including some glucose. It attacks carboxymethylcellulose and xylan at approximately the same rates. Its activity on carboxymethylcellulose is much higher than that of the isolated C. acetobutylicum cellulosome. It also extensively converts lichenan to oligosaccharides of intermediate size and attacks Avicel to a limited extent. The enzyme has an optimal temperature in a 10-min assay of 55 degrees C and an optimal pH of 5.0.

Progress in the overall understanding of typhoid fever: implications for vaccine development.

INTRODUCTION: Typhoid fever continues to have a substantial impact on human health, especially in Asia and sub-Saharan Africa. Access to safe water, and adequate sanitation and hygiene remain the cornerstone of prevention, but these are not widely available in many impoverished settings. The emergence of antibiotic resistance affects typhoid treatment and adds urgency to typhoid control efforts. Vaccines provide opportunities to prevent and control typhoid fever in endemic settings. AREAS COVERED: Literature search was performed looking for evidence concerning the global burden of typhoid and strategies for the prevention and treatment of typhoid fever. Cost of illness, available typhoid and paratyphoid vaccines and cost-effectiveness were also reviewed. The objective was to provide a critical overview of typhoid fever, in order to assess the current understanding and potential future directions for typhoid treatment and control. EXPERT COMMENTARY: Our understanding of typhoid burden and methods of prevention has grown over recent years. However, typhoid fever still has a significant impact on health in low and middle-income countries. Introduction of typhoid conjugate vaccines to the immunization schedule is expected to make a major contribution to control of typhoid fever in endemic countries, although vaccination alone is unlikely to eliminate the disease.

Mechanism of cellulose hydrolysis by inverting GH8 endoglucanases: a QM/MM metadynamics study.

A detailed understanding of the catalytic strategy of cellulases is key to finding alternative ways to hydrolyze cellulose to mono-, di-, and oligosaccharides. Endoglucanases from glycoside hydrolase family 8 (GH8) catalyze the hydrolysis of beta-1,4-glycosidic bonds in cellulose by an inverting mechanism believed to involve a oxacarbenium ion-like transition state (TS) with a boat-type conformation of the glucosyl unit in subsite -1. In this work, hydrolysis by Clostridium thermocellum endo-1,4-glucanase A was computationally simulated with quantum mechanics/molecular mechanics metadynamics based on density functional theory. Our calculations show that the glucosyl residue in subsite -1 in the Michaelis complex is in a distorted 2SO/2,5B ring conformation, agreeing well with its crystal structure. In addition, our simulations capture the cationic oxacarbenium ion-like character of the TS with a partially formed double bond between the ring oxygen and C5' carbon atoms. They also provide previously unknown structural information of important states along the reaction pathway. The simulations clearly show for the first time in GH8 members that the TS features a boat-type conformation of the glucosyl unit in subsite -1. The overall catalytic mechanism follows a DN*AN-like mechanism and a beta-2SO-->2,5B [TS]-->alpha-5S1 conformational itinerary along the reaction coordinate, consistent with the anti-periplanar lone pair hypothesis. Because of the structural similarities and sequence homology among all GH8 members, our results can be extended to all GH8 cellulases, xylanases, and other endoglucanases. In addition, we provide evidence supporting the role of Asp278 as the catalytic proton acceptor (general base) for GH8a subfamily members.

A Gibbs free energy correlation for automated docking of carbohydrates.

Thermodynamic information can be inferred from static atomic configurations. To model the thermodynamics of carbohydrate binding to proteins accurately, a large binding data set has been assembled from the literature. The data set contains information from 262 unique protein-carbohydrate crystal structures for which experimental binding information is known. Hydrogen atoms were added to the structures and training conformations were generated with the automated docking program AutoDock 3.06, resulting in a training set of 225,920 all-atom conformations. In all, 288 formulations of the AutoDock 3.0 free energy model were trained against the data set, testing each of four alternate methods of computing the van der Waals, solvation, and hydrogen-bonding energetic components. The van der Waals parameters from AutoDock 1 produced the lowest errors, and an entropic model derived from statistical mechanics produced the only models with five physically and statistically significant coefficients. Eight models predict the Gibbs free energy of binding with an error of less than 40% of the error of any similar models previously published.

Theory and computation show that Asp463 is the catalytic proton donor in human endoplasmic reticulum alpha-(1-->2)-mannosidase I.

It has been difficult to identify the proton donor and nucleophilic assistant/base of endoplasmic reticulum alpha-(1-->2)-mannosidase I, a member of glycoside hydrolase Family 47, which cleaves the glycosidic bond between two alpha-(1-->2)-linked mannosyl residues by the inverting mechanism, trimming Man(9)GlcNAc(2) to Man(8)GlcNAc(2) isomer B. Part of the difficulty is caused by the enzyme's use of a water molecule to transmit the proton that attacks the glycosidic oxygen atom. We earlier used automated docking to conclusively determine that Glu435 in the yeast enzyme (Glu599 in the corresponding human enzyme) is the nucleophilic assistant. The commonly accepted proton donor has been Glu330 in the human enzyme (Glu132 in the yeast enzyme). However, for theoretical reasons this conclusion is untenable. Theory, automated docking of alpha-d-(3)S(1)-mannopyranosyl-(1-->2)-alpha-d-(4)C(1)-mannopyranose and water molecules associated with candidate proton donors, and estimation of dissociation constants of the latter have shown that the true proton donor is Asp463 in the human enzyme (Asp275 in the yeast enzyme).

Computational analyses of the conformational itinerary along the reaction pathway of GH94 cellobiose phosphorylase.

GH94 cellobiose phosphorylase (CBP) catalyzes the phosphorolysis of cellobiose into alpha-D-glucose 1-phosphate (G1P) and D-glucose with inversion of anomeric configuration. The complex crystal structure of CBP from Cellvibrio gilvus had previously been determined; glycerol, glucose, and phosphate are bound to subsites -1, +1, and the anion binding site, respectively. We performed computational analyses to elucidate the conformational itinerary along the reaction pathway of this enzyme. autodock was used to dock cellobiose with its glycon glucosyl residue in various conformations and with its aglycon glucosyl residue in the low-energy 4C1 conformer. An oxocarbenium ion-like glucose molecule mimicking the transition state was also docked. Based on the clustering analysis, docked energies, and comparison with the crystallographic ligands, we conclude that the reaction proceeds from 1S3 as the pre-transition state conformer (Michaelis complex) via E3 as the transition state candidate to 4C1 as the G1P product conformer. The predicted reaction pathway of the inverting phosphorylase is similar to that proposed for the first-half glycosylation reaction of retaining cellulases, but is different from those for inverting cellulases. NAMD was used to simulate molecular dynamics of the enzyme. The 1S3 pre-transition state conformer is highly stable compared with other conformers, and a conformational change from 4C1 to 1,4B was observed.

The fate of beta-D-mannopyranose after its formation by endoplasmic reticulum alpha-(1-->2)-mannosidase I catalysis.

The automated docking program AutoDock was used to dock all 38 characteristic beta-D-mannopyranose ring conformers into the active site of the yeast endoplasmic reticulum alpha-(1-->2)-mannosidase I, a Family 47 glycoside hydrolase that converts Man9GlcNAc2 to Man8GlcNAc2. The subject of this work is to establish the conformational pathway that allows the cleaved glycon product to leave the enzyme active site and eventually reach the ground-state conformation. Twelve of the 38 conformers optimally dock in the active site where the inhibitors 1-deoxymannonojirimycin and kifunensine are found in enzyme crystal structures. A further 23 optimally dock in a second site on the side of the active-site well, while three dock outside the active-site cavity. It appears, through analysis of the internal energies of different ring conformations, of intermolecular energies between the ligands and enzyme, and of forces exerted on the ligands by the enzyme, that beta-D-mannopyranose follows the path 3E-->1C4-->1H2-->B2,5 before being expelled by the enzyme. The highly conserved second site that strongly binds beta-D-mannopyranose-4C1 may exist to prevent competitive inhibition by the product, and is worthy of further investigation.

Ruminal fermentation of propylene glycol and glycerol.

Bovine rumen fluid was fermented anaerobically with 25 mM R-propylene glycol, S-propylene glycol, or glycerol added. After 24 h, all of the propylene glycol enantiomers and approximately 80% of the glycerol were metabolized. Acetate, propionate, butyrate, valerate, and caproate concentrations, in decreasing order, all increased with incubation time. Addition of any of the three substrates somewhat decreased acetate formation, while addition of either propylene glycol increased propionate formation but decreased that of butyrate. R- and S-propylene glycol did not differ significantly in either their rates of disappearance or the products formed when they were added to the fermentation medium. Fermentations of rumen fluid containing propylene glycol emitted the sulfur-containing gases 1-propanethiol, 1-(methylthio)propane, methylthiirane, 2,4-dimethylthiophene, 1-(methylthio)-1-propanethiol, dipropyl disulfide, 1-(propylthio)-1-propanethiol, dipropyl trisulfide, 3,5-diethyl-1,2,4-trithiolane, 2-ethyl-1,3-dithiane, and 2,4,6-triethyl-1,3,5-trithiane. Metabolic pathways that yield each of these gases are proposed. The sulfur-containing gases produced during propylene glycol fermentation in the rumen may contribute to the toxic effects seen in cattle when high doses are administered for therapeutic purposes.

Comparing programs for rigid-body multiple structural superposition of proteins.

Different programs and methods were employed to superimpose protein structures, using members of four very different protein families as test subjects, and the results of these efforts were compared. Algorithms based on human identification of key amino acid residues on which to base the superpositions were nearly always more successful than programs that used automated techniques to identify key residues. Among those programs automatically identifying key residues, MASS could not superimpose all members of some families, but was very efficient with other families. MODELLER, MultiProt, and STAMP had varying levels of success. A genetic algorithm program written for this project did not improve superpositions when results from neighbor-joining and pseudostar algorithms were used as its starting cases, but it always improved superpositions obained by MODELLER and STAMP. A program entitled PyMSS is presented that includes three superposition algorithms featuring human interaction.

Problem-based learning biotechnology courses in chemical engineering.

We have developed a series of upper undergraduate/graduate lecture and laboratory courses on biotechnological topics to supplement existing biochemical engineering, bioseparations, and biomedical engineering lecture courses. The laboratory courses are based on problem-based learning techniques, featuring two- and three-person teams, journaling, and performance rubrics for guidance and assessment. Participants initially have found them to be difficult, since they had little experience with problem-based learning. To increase enrollment, we are combining the laboratory courses into 2-credit groupings and allowing students to substitute one of them for the second of our 2-credit chemical engineering unit operations laboratory courses.

Docking studies on glycoside hydrolase Family 47 endoplasmic reticulum alpha-(1-->2)-mannosidase I to elucidate the pathway to the substrate transition state.

Alpha-(1-->2)-mannosidase I from the endoplasmic reticulum (ERManI), a Family 47 glycoside hydrolase, is a key enzyme in the N-glycan synthesis pathway. Catalytic-domain crystal structures of yeast and human ERMan1s have been determined, the former with a hydrolytic product and the latter without ligands, with the inhibitors 1-deoxymannojirimycin and kifunensine, and with a thiodisaccharide substrate analog. Both inhibitors were bound at the base of the funnel-shaped active site as the unusual 1C4 conformer, while the substrate analog glycon is a 3S1 conformer. In the current study, AutoDock was used to dock alpha-D-mannopyranosyl-(1-->2)-alpha-D-mannopyranose with its glycon in chair (1C4,4C1), half-chair (3H2,3H4,4H3), skew-boat (OS2,3S1,5S1), boat (2,5B,3,OB,B1,4,B2,5), and envelope (3E,4E,E3,E4) conformations into the yeast ERManI active site. Both docked energies and forces on docked ligand atoms were calculated to determine how the ligand distorts to the transition state. From these, we can conclude that (1) both 1C4 and OS2 can be the starting conformers; (2) the most likely binding pathway is 1C4-->3H2-->OS2-->3,OB-->3S1-->3E; (3) the transition state is likely to be close to a 3E conformation.