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Expression und funktionelle Charakterisierung des Schlüsselenzyms der Sialinsäurebiosynthese, UDP-GlcNAc-2-Epimerase/ManNAc-KinaseBlume, Astrid. January 2003 (has links)
Berlin, Freie Universiẗat, Diss., 2003. / Dateiformat: zip, Dateien im PDF-Format.
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Mechanistic studies on ADP-L-glycero-D-manno-heptose 6-epimerase and UDP-N-acetylglucosamine 5-inverting 4,6-dehydrataseMorrison, James P. 05 1900 (has links)
ADP-L-glycero-D-manno-heptose 6-epimerase (HldD) catalyzes the inversion of configuration at C-6" of the heptose moiety of ADP-D-glycero-D-manno-heptose and ADP-L-glycero-D-manno-heptose. H1dD operates in the biosynthesis of L-glycero-D-manno-heptose, a conserved sugar in the core region of lipopolysaccharide (LPS) of Gram-negative bacteria. This work supports a direct redox mechanism whereby H1dD uses its tightly bound NADP+ to oxidize the substrate at C-6", generating a ketone intermediate. Reduction from the opposite face generates the epimeric product. An analog of the ketone intermediate, ADP-ß-D-manno-hexodialdose 8, was shown to undergo dismutation giving equal amounts of ADP-mannose 9and ADP-mannuronate 10. Observation of transient NADPH during dismutation established participation of the tightly bound cofactor.
Further studies address how HldD is able to access both faces of the ketone intermediate with correct alignment of NADPH, the ketone intermediate, and a catalytic acid/base residue. It is proposed that Escherichia coli K-12 HldD contains two catalytic acid/base residues, tyrosine 140 and lysine 178, each of which facilitates redox chemistry on opposite faces of the ketone intermediate. The ketone intermediate may access either base via rotation about the C-5"/C-6" bond. The observation that two single mutants, Y140F and K178M, have severely compromised epimerase activities, yet retain dismutase activity, supports this hypothesis.
UDP-N-acetylglucosamine 5-inverting 4,6-dehydratase (PseB) is a unique sugar nucleotide dehydratase that inverts the C-5" stereocentre during conversion of UDP-N-acetylglucosamine to UDP-2-acetyl-2,6-dideoxy-ß-L-arabino-4-hexulose. PseB catalyses the first step in the biosynthesis of pseudaminic acid, which is found as a post-translational modification on the flagellin of Campylobacter jejuni and Helicobacter pylon. PseB uses its tightly bound NADP+ to oxidize UDP-G1cNAc at C-4", enabling dehydration. The a,ß unsaturated ketone intermediate thus generated is reduced by delivery of a hydride from NADPH to C-6", and a proton to C-5". Consistent with this mechanism, a solvent derived deuterium becomes incorporated into the C-5" position of product during catalysis in D20. Likewise, PseB catalyzes solvent isotope exchange into the H5" position of the product, and theelimination of HF from UDP-6-deoxy-6-fluoro-G1cNAc 23. Mutants of the putative catalytic residues aspartate 126, lysine 127 and tyrosine 135 have severely compromised dehydratase, solvent isotope exchange, and HF elimination activities.
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Mechanistic studies on ADP-L-glycero-D-manno-heptose 6-epimerase and UDP-N-acetylglucosamine 5-inverting 4,6-dehydrataseMorrison, James P. 05 1900 (has links)
ADP-L-glycero-D-manno-heptose 6-epimerase (HldD) catalyzes the inversion of configuration at C-6" of the heptose moiety of ADP-D-glycero-D-manno-heptose and ADP-L-glycero-D-manno-heptose. H1dD operates in the biosynthesis of L-glycero-D-manno-heptose, a conserved sugar in the core region of lipopolysaccharide (LPS) of Gram-negative bacteria. This work supports a direct redox mechanism whereby H1dD uses its tightly bound NADP+ to oxidize the substrate at C-6", generating a ketone intermediate. Reduction from the opposite face generates the epimeric product. An analog of the ketone intermediate, ADP-ß-D-manno-hexodialdose 8, was shown to undergo dismutation giving equal amounts of ADP-mannose 9and ADP-mannuronate 10. Observation of transient NADPH during dismutation established participation of the tightly bound cofactor.
Further studies address how HldD is able to access both faces of the ketone intermediate with correct alignment of NADPH, the ketone intermediate, and a catalytic acid/base residue. It is proposed that Escherichia coli K-12 HldD contains two catalytic acid/base residues, tyrosine 140 and lysine 178, each of which facilitates redox chemistry on opposite faces of the ketone intermediate. The ketone intermediate may access either base via rotation about the C-5"/C-6" bond. The observation that two single mutants, Y140F and K178M, have severely compromised epimerase activities, yet retain dismutase activity, supports this hypothesis.
UDP-N-acetylglucosamine 5-inverting 4,6-dehydratase (PseB) is a unique sugar nucleotide dehydratase that inverts the C-5" stereocentre during conversion of UDP-N-acetylglucosamine to UDP-2-acetyl-2,6-dideoxy-ß-L-arabino-4-hexulose. PseB catalyses the first step in the biosynthesis of pseudaminic acid, which is found as a post-translational modification on the flagellin of Campylobacter jejuni and Helicobacter pylon. PseB uses its tightly bound NADP+ to oxidize UDP-G1cNAc at C-4", enabling dehydration. The a,ß unsaturated ketone intermediate thus generated is reduced by delivery of a hydride from NADPH to C-6", and a proton to C-5". Consistent with this mechanism, a solvent derived deuterium becomes incorporated into the C-5" position of product during catalysis in D20. Likewise, PseB catalyzes solvent isotope exchange into the H5" position of the product, and theelimination of HF from UDP-6-deoxy-6-fluoro-G1cNAc 23. Mutants of the putative catalytic residues aspartate 126, lysine 127 and tyrosine 135 have severely compromised dehydratase, solvent isotope exchange, and HF elimination activities.
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Mechanistic studies on ADP-L-glycero-D-manno-heptose 6-epimerase and UDP-N-acetylglucosamine 5-inverting 4,6-dehydrataseMorrison, James P. 05 1900 (has links)
ADP-L-glycero-D-manno-heptose 6-epimerase (HldD) catalyzes the inversion of configuration at C-6" of the heptose moiety of ADP-D-glycero-D-manno-heptose and ADP-L-glycero-D-manno-heptose. H1dD operates in the biosynthesis of L-glycero-D-manno-heptose, a conserved sugar in the core region of lipopolysaccharide (LPS) of Gram-negative bacteria. This work supports a direct redox mechanism whereby H1dD uses its tightly bound NADP+ to oxidize the substrate at C-6", generating a ketone intermediate. Reduction from the opposite face generates the epimeric product. An analog of the ketone intermediate, ADP-ß-D-manno-hexodialdose 8, was shown to undergo dismutation giving equal amounts of ADP-mannose 9and ADP-mannuronate 10. Observation of transient NADPH during dismutation established participation of the tightly bound cofactor.
Further studies address how HldD is able to access both faces of the ketone intermediate with correct alignment of NADPH, the ketone intermediate, and a catalytic acid/base residue. It is proposed that Escherichia coli K-12 HldD contains two catalytic acid/base residues, tyrosine 140 and lysine 178, each of which facilitates redox chemistry on opposite faces of the ketone intermediate. The ketone intermediate may access either base via rotation about the C-5"/C-6" bond. The observation that two single mutants, Y140F and K178M, have severely compromised epimerase activities, yet retain dismutase activity, supports this hypothesis.
UDP-N-acetylglucosamine 5-inverting 4,6-dehydratase (PseB) is a unique sugar nucleotide dehydratase that inverts the C-5" stereocentre during conversion of UDP-N-acetylglucosamine to UDP-2-acetyl-2,6-dideoxy-ß-L-arabino-4-hexulose. PseB catalyses the first step in the biosynthesis of pseudaminic acid, which is found as a post-translational modification on the flagellin of Campylobacter jejuni and Helicobacter pylon. PseB uses its tightly bound NADP+ to oxidize UDP-G1cNAc at C-4", enabling dehydration. The a,ß unsaturated ketone intermediate thus generated is reduced by delivery of a hydride from NADPH to C-6", and a proton to C-5". Consistent with this mechanism, a solvent derived deuterium becomes incorporated into the C-5" position of product during catalysis in D20. Likewise, PseB catalyzes solvent isotope exchange into the H5" position of the product, and theelimination of HF from UDP-6-deoxy-6-fluoro-G1cNAc 23. Mutants of the putative catalytic residues aspartate 126, lysine 127 and tyrosine 135 have severely compromised dehydratase, solvent isotope exchange, and HF elimination activities. / Science, Faculty of / Chemistry, Department of / Graduate
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Molecular studies of galactan biosynthesis in red algaeHector, Stanton Bevan Ernest 12 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Sulfated galactans (agarans and carrageenans) are accumulated in the cell wall of various red
algae (Rhodophyta) species. These polysaccharides are of commercial importance in the food,
pharmaceutical and biotechnology industries due to their unique physicochemical properties.
Although having received significant research attention over the last 20 years, events
regarding their biosynthesis have not been elucidated. Aiming for the identification of
galactosyltransferase (GalT) genes involved in sulfated galactan biosynthesis, cDNA
expression libraries were constructed from the prolific agar-producing South African red
seaweed Gelidium pristoides (Turner) Kützing and screened by functional complementation
of UDP-galactose 4-epimerase deficient mutants (E. coli and S. cerevisiae). Regretfully, no
GalTs were identified. The study however yielded the first UGE enzyme described for a red
seaweed. Southern hybridization indicated the presence of two UGE copies and confirmed the
gene originated from G. pristoides. Bioinformatic analysis of G. pristoides UGE shows amino
acid sequence homology to known UGEs from various organisms. The enzyme was shown to
be functional in E. coli crude extracts and showed affinity for UDP-D-galactose, similar to
other UDP-galactose 4-epimerases. Further, the isolated G. pristoides UGE (GpUGE) was
biochemically characterized and its kinetic parameters determined. We found that there was
no kinetic difference between this enzyme and previously described UGE enzymes except
enhanced activity in the presence of exogenously added NAD+.
The UDP-galactose 4-epimerase (UDP-glucose 4-epimerase, UGE, EC 5.1.3.2) is an essential
Leloir pathway enzyme facilitating the catalytic inter-conversion between UDP-D-glucose
and UDP-D-galactose. UDP-D-galactose is the nucleotide sugar required by
galactosyltransferases for the production of red algae sulfated galactans. UGE is suspected as
being responsible for supplying UDP-D-galactose for the synthesis of sulfated galactans. In
planta monitoring of GpUGE transcript levels with respect to dark and light cycling indicated
high expression of the enzyme at night, while expression diminished during the day. The
occurrence of increased nocturnal UGE expression correlates with floridean starch breakdown
at night. Evidence for hydrolysis of floridean starch is also reflected in obtained G. pristoides
transcriptome sequence data. In red algae, floridean starch degradation coincides with sulfated
galactan production. The detection of starch hydrolysis enzyme transcripts alongside
increased expression of GpUGE suggests the enzyme plays a role in supplying UDP-Dgalactose
for sulfated galactan production. As far as we know, this the first report of
sequencing and biochemical characterization of a UGE from red seaweed.
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Investigation of small molecules binding to UDP-galactose 4'-epimerase : - A validated drug target for <em>Trypanosoma brucei</em>, the parasite responsible for African Sleeping Sickness.Jinnelöv, Anders January 2009 (has links)
No description available.
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Investigation of small molecules binding to UDP-galactose 4'-epimerase : A validated drug target for Trypanosoma brucei, the parasite responsible for African Sleeping Sickness.Jinnelöv, Anders January 2009 (has links)
African sleeping sickness is a parasitic infection spread by the protozoan parasite Trypanosoma brucei, and drugs used today are toxic and painful. Galactose metabolism is essential for the survival of T. brucei and without a functional UDP galactose 4’ epimerase (GalE) galactose starvation occurs and cell death will follow. In this Master thesis project two assays observing binding of small molecules to TbGalE has been investigated in attempt to establish an assay that in the future could be used for screening for drugs. TbGalE was biotinylated through the Pinpoint Xa vector and expressed in E. coli cells. The protein was successfully immobilized to a Streptavidin chip for Surface Plasmon Resonance experiments and the binding of the substrates UDP-galactose and UDP-glucose was observed. Unfortunately, the assay was not optimal for screening due to low signal response. However, the established protocol for expressing biotinylated proteins that bind to Streptavidin surfaces could be used in further experiments with TbGalE and other drug targets for African sleeping sickness. The fluorescent sugar nucleotide analogue UDPAmNS, which is a known inhibitor for E. coli GalE, was synthesised and purified and then used to establish a displacement assay. IC50 of UDPAmNS against TbGalE was determined and a synergic effect in fluorescence between the protein and the inhibitor was proven. Further, evidence for a reduction in fluorescence by displacing UDPAmNS with UDP was obtained. This reduction in fluorescence was also shown by a predicted cofactor inhibitor. The IC50 against TbGalE for this compound was determined before the displacement assay, which showed that the cofactor inhibitor, at least partly, binds to the active site of TbGalE. The UDPAmNS displacement assay could have the potential of becoming a robust screening assay for TbGalE, in the effort to find a better drug for African sleeping sickness.
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Dissecting tunicamycin biosynthesis : a potent carbohydrate processing enzyme inhibitorWyszynski, Filip Jan January 2010 (has links)
Tunicamycin nucleoside antibiotics were the first known to target the formation of peptidoglycan precursor lipid I in bacterial cell wall biosynthesis. They have also been used extensively as inhibitors of protein N-glycosylation in eukaryotes, blocking the biogenesis of early intermediate dolichyl-pyrophosphoryl-N-acetylglucosamine. Despite their unusual structures and useful biological properties, little is known about their biosynthesis. Elucidating the metabolic pathway of tunicamycins and gaining an understanding of the enzymes involved in key bond forming processes would not only be of great academic value in itself, it would also unlock a comprehensive toolbox of biosynthetic machinery for the production of tunicamycin analogues which have the potential to act as novel therapeutic antibiotics or as specific inhibitors of medicinally important NDP-dependent glycosyltransferases. I – Cloning the tunicamycin biosynthetic gene cluster. We report identification of the tunicamycin biosynthetic genes in Streptomyces chartreusis following genome sequencing and a chemically-guided strategy for in silico genome mining that allowed rapid identification and unification of an operon fractured across contigs. Heterologous expression established a likely minimal gene set necessary for antibiotic production, from which a detailed metabolic pathway for tunicamycin biosynthesis is proposed. II – Natural product isolation and degradation. We have developed efficient methods for the isolation of tunicamycins from liquid culture in preparative quantities. A subsequent relay synthesis furnished advanced biosynthetic intermediates for use as precursors in the production of tunicamycin analogues and as substrates for the in vitro characterisation of individual Tun enzymes. III – Functional characterisation of tun gene products. Individual tun gene products were over-expressed and purified from recombinant E. coli hosts, allowing in vitro functional studies to take place. An NMR assay of biosynthetic enzyme TunF showed it acted as a UDP-GlcNAc-4-epimerase. Putative glycosyltransferase TunD showed hydrolytic activity towards substrate UDP-GlcNAc but failed to accept to the expected natural acceptor substrate, providing unexpected insights into the ordering of biosynthetic events in the tunicamycin pathway. Initial studies into the over-expression of the putative sugar N-deacetylase TunE were also described. IV – Towards synthesis of tunicamycin fragments. Investigations into a novel synthesis of D-galactosamine – a structural motif within tunicamycin – led to the unexpected observation of inverted regioselectivity upon RhII-catalysed C-H insertion of a D-mannose-derived sulfamate. This was the first example of N-insertion at the beta- rather than gamma-C-H based on conformation alone and warranted further investigation. The X-ray structure of a key sulfamate precursor offered valuable insights as to the source of this unique selectivity.
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Transcriptional Regulation And The Role Of Galactose Metabolism In The Virulence Of Candida AlbicansSingh, Vijender 03 1900 (has links)
Candida albicans, a commensal of gastrointestinal and uro-vaginal tract can cause superficial as well as life threatening disseminated infections under conditions of lowered immunity of the host such as HIV infection, drug induced immune suppression [given during organ transplantation to prevent rejection] and radiation therapy [head and neck
cancer patients] (Odds, 1988; Fidel and Sobel, 1996). Candida albicans shows a range of morphologies, it can switch from budding yeast morphology to pseudohyphae (chains of elongated cells with visible constrictions at the sites of septa) and hyphae (linear filaments without visible constrictions at the septa) (Mitchell, 1998). The various factors that contribute to its virulence include its ability to undergo yeast to hyphal transition,
formation of biofilms, adhesion and secretion of aspartyl proteinases. Hyphae are considered to be involved in invasive growth as they are frequently identified in infected tissues and strains defective in morphological transition (yeast to hyphal) are avirulent (Leberer et al., 1996; Lo et al., 1997; Stoldt et al., 1997). Morphological switching is not only necessary for successful establishment of infection but important for evading
components host defense system like macrophages or dendritic cells. A network of signaling pathways that operate in C. albicans continuously assess the nutrient availability, cell density and other environmental conditions. The integrated output of these pathways determine the response of C. albicans under given set of environmental/media conditions and eventually determines the gene expression and morphogenic transition (Liu., 2001). C. albicans utilizes at least two major signaling pathways besides others for
regulating the morphological transition. One of these two pathways uses Cph1 as
transcription factor and is the homolog of Ste12 in S. cerevisiae which is shown to be
involved in Pseudohyphal growth and mating. The other pathway includes Efg1
(homolog of Phd1 in S. cerevisiae) as transcription factor.
Biofilm formation by Candida species is an important virulence factor and has
gained considerable interest recently as these specialized survival structures are found in implanted devices such as indwelling catheters and prosthetic heart valves (Hawser and Douglas, 1994; Douglas, 2003). These biofilms lead to the failure of implants besides providing multiple drug resistance (Baillie and Douglas, 1999).
A better understanding of the C. albicans interaction with the host at the site of
infection and with the components of immune system will help in identifying new
potential drug targets.
(a) Genome wide expression profile of Candida albicans from patient samples and
characterization of CaRPB4/7:
To get a better insight in C. albicans response at the site of infection we were
interested in mapping the expression profile of Candida albicans in active state of human
infections. Patients suffering from head and neck cancer undergoing radiation therapy
have high risk of C. albicans infection. We identified five such patients with heavy oral thrush infections and C. albicans samples were collected from them. Candida albicans was confirmed in these samples by various microbiological tests following which the samples were used for RNA isolation. The whole genome expression analysis leads to the identification of 188 up regulated and 88 down regulated genes in patient samples. Our data analysis revealed that Protein Kinase A pathway and many downstream genes of the same were differentially expressed. Analysis of saliva (saliva is known for antifungal and
antibacterial activity) from these patients showed that unlike healthy individuals, the
patient saliva favours yeast to hyphal transition of C. albicans cells. This might be a reason for high risk of infection. A major class of upregulated genes is found to be functionally involved in transcription which includes some RNA polymeraseII and III
subunits. CaRPB4, the forth largest subunit of RNA polymeraseII, was found to be
upregulated in patient samples. RPB4 has been shown to form sub complex with RPB7,
the seventh largest subunit of RNA polymeraseII, and both subunits are known to play a role in a variety of stress conditions and pseudohyphal development in Saccharomyces cerevisiae. We characterized the CaRPB4 and CaRPB7 (homolog in Candida albicans) for their ability to complement their S. cerevisiae counterparts. CaRPB4 and CaRPB7 were able to complement majority of the phenotypes associated with these subunits in S. cerevisiae. Overexpression of CaRPB7 in S. cerevisiae enhances pseudohyphal growth. Considering the high degree of conservation of signaling pathways between S. cerevisiae and C. albicans it can be speculated that CaRPB7 might be involved in pseudohyphal development in C. albicans. We found that over expression of CaRPB4 in Candida albicans shows enhanced agar invasive growth which can be thought analogous to tissue invasion in host and hence might contribute for establishment of infection. This suggests that both the RNA polII subunits have a role to play in the virulence of C. albicans.
(b) Characterization of UDP-Galactose 4-Epimerase (GAL10) from Candida albicans and their role in virulence.
Enzyme UDP-Galactose-4-Epimerase [GAL10] is responsible for conversion of UDP-galactose to UDP-glucose which then gets metabolized by the cells through glycolysis and TCA cycle. The enzyme catalyzes a reversible reaction and can convert glucose to galactose in the absence of galactose as shown in Trypanosoma brucei and also
involved in its virulence. In this study, we have identified the functional homolog of
GAL10 in Candida albicans. S. cerevisiae and C. albicans GAL10 homologs are similar in their domainal organization as the proteins have a mutarotase and an epimerase domain. The former is responsible for conversion of ゚-D-galactose to a-D-galactose and the latter for epimerization of UDP-galactose to UDP-glucose. The synteny of galactose metabolizing structural genes is conserved among some fungi. To study the importance of CaGAL10 we generated deletion mutant of the gene in C. albicans. Our studies show that CaGAL10 [C. albicans GAL10] is involved in cell wall organization and in oxidative stress response. The mutant strain of GAL10 is hyperfilamentous in Lee’s and spider medium and the biofilm formed is morphologically different from the wild type strain. These set of results suggests that CaGAL10 plays an important role in organization/integrity of cell wall in C. albicans and speculate that it might be involved in virulence.
(c) Study of Candida albicans-macrophage interaction and identification of
transcriptional regulator of genes encoding proteins of translation machinery:
Macrophages serve as the effector cells of cell mediated immunity in the control of infections. They are considered to be important for resistance to muco-cutaneous and systemic candidiasis. Our studies were aimed at understanding the response of Candida albicans cells to the presence of macrophages for extended period of time. The response was monitored using microarrays. Specifically genes involved in galactose, protein and lipid metabolism and stress response undergo concerted changes in their transcript levels. We analyzed the promoters of coregulated genes to identify common DNA elements present in them which might be involved in their transcriptional regulation. Promoter analysis of differentially expressed genes revealed presence of CPH1 and EFG1 transcription factor binding sites. Besides identifying CPH1 and EFG1 Binding sites, we identified two novel DNA elements in promoters of coregulated gene. A conserved motif TGAAAAGGAAG was identified in the promoters of genes involved in energy generation. Another 18 mer consensus palindromic sequence
TAGGGCTNTAGCCCTAAT was identified in the promoters of about 48 genes. Majority of these genes encode ribosomal proteins. With the help of techniques like EMSA (Electophoretic Mobility Shift Assay) and south-western we had shown the presence of a protein of ~66 KDa molecular weight binding to the sequence with high specificity.
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Advancing understanding of secondary cell wall polymer binding and synthesis in S-layers of Gram-Positive bacteriaLegg, Max 21 April 2022 (has links)
Self-assembling protein surface layers (S-layers) are ubiquitous prokaryotic cell-surface structures involved in structural maintenance, nutrient diffusion, host adhesion, virulence, and many additional processes, which makes them appealing targets for therapeutics and biotechnological applications, including live vaccines, liposome drug delivery and biosensors. Unlocking this potential requires expanding our understanding of S-layer properties, especially the details of surface-attachment.
S-layers of Gram-positive bacteria often are attached through the interaction of specialized S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Characterization of this interaction in the Gram-positive model organism Paenibacillus alvei CCM 2051T reveals that, remarkably, binding-site switches can occur between two distinct SLH-domain SCWP receptor-site grooves in the S-layer protein SpaA, possibly as part of a mechanism to alleviate strain in the S-layer. To date, however, analysis of this novel mechanism has been limited to the terminal SCWP monosaccharide and the internal SCWP repeat disaccharide ligand analogues, leaving open the role of subsequent SCWP sugar residues in binding, as well as whether the two receptor sites are also suited to accommodate longer SCWP ligands that better approximate the biological target at the surface of P. alvei.
To address this, the objective of this work aims to uncover and characterize the details of the SpaA SLH-domain (SpaASLH¬) SCWP-interaction by determining the co-crystal structures of SpaASLH¬, and single (SpaASLH/G109A) and the corresponding double (SpaASLH/G46A/G109A) mutants in complex with synthetic terminal disaccharide and trisaccharide analogues of the P. alvei CCM 2051T SCWP target. These structural characterizations have been supplemented with disaccharide and trisaccharide binding data, which was obtained through thermodynamic ITC analyses carried out by collaborators.
The co-crystal structures of P. alvei SpaASLH with synthetic, terminal SCWP disaccharide and trisaccharide analogues, together with previously published monosaccharide-bound SpaASLH structures, reveal that while the SLH trimer accommodates longer biologically relevant SCWP ligands within both its primary (G2) and secondary (G1) binding sites, the terminal pyruvylated ManNAc moiety serves as the nearly-exclusive SCWP anchoring point. Binding is accompanied by displacement of a flexible loop adjacent to the receptor site that enhances the complementarity between protein and ligand, including electrostatic complementarity with the terminal pyruvate moiety. Remarkably, binding of the pyruvylated monosaccharide SCWP fragment alone is sufficient to cause rearrangement of the receptor binding sites in a manner necessary to accommodate longer SCWP fragments. The observation of multiple conformations for longer oligosaccharides bound to the protein, together with the demonstrated functionality of two of the three SCWP receptor binding sites, reveals how the SpaASLH-SCWP interaction has evolved to accommodate longer SCWP ligands and alleviate the strain inherent to bacterial S-layer adhesion during growth and division.
In addition, to further clarify the steps involved in SCWP biosynthesis, we present a crystal structure of the unliganded UDP-GlcNAc 2-epimerase enzyme MnaA, which catalyzes the interconversion of UDP-GlcNAc into UDP-ManNAc—an essential building block of the P. alvei SCWP target. The P. alvei MnaA epimerase adopts a GT-B fold that is consistent with the architecture of previously published structures of other bacterial non-hydrolyzing UDP-GlcNAc 2-epimerase enzymes for which substrate binding is observed in the cleft located between the two domains. Characterization of this structure, coupled with an analysis of the sequence of the MnaA protein, reveals the presence of conserved residues that define the catalytic and allosteric sites in homologous enzymes from different organisms. These residues are positioned to accommodate substrate within the MnaA binding cleft in much the same manner as the published enzyme homologues, suggesting that allosteric regulation as a mechanism for enzyme regulation is conserved in P. alvei MnaA.
These investigations are part of a greater effort toward understanding SLH domain-mediated SCWP-interactions in Gram-positive organisms, and provide insight into the structure and putative function of this SCWP biosynthetic enzyme. By understanding these processes, this knowledge may contribute to providing a platform for the rational design of Gram-positive inhibitors. Such inhibitors could selectively target, for example, the bacterial S-layer SCWP-binding interaction, or perhaps the essential biosynthetic enzymes involved in producing the exclusive targets that these S-layer proteins recognize and bind, and would thus represent a new class of antimicrobial therapeutics. / Graduate
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