Ferulic acid is a common constituent of the plant cell-wall matrix where it decorates and can crosslink mainly arabinoxylans to provide structural reinforcement. Microbial feruloyl esterases (FAEs) specialize in catalyzing hydrolysis of the ester bonds between phenolic acids and sugar residues in plant cell-wall polysaccharides such as arabinoxylan to release cinnamoyl compounds. Feruloyl esterases from lactic acid bacteria (LAB) have been highlighted as interesting enzymes for their potential applications in the food and pharmaceutical industries; however, there are few studies on the activity and structure of FAEs of LAB origin. Here, we report the crystal structure and biochemical characterization of a feruloyl esterase (LbFAE) from Lentilactobacillus buchneri, a LAB strain that has been used as a silage additive. The LbFAE structure was determined in the absence and presence of product (FA) and reveals a new type of homodimer association not previously observed for fungal or bacterial FAEs. The two subunits associate to restrict access to the active site such that only single FA chains attached to arabinoxylan can be accommodated, an arrangement that excludes access to FA cross-links between arabinoxylan chains. This narrow specificity is further corroborated by the observation that no FA dimers are produced, only FA, when feruloylated arabinoxylan is used as substrate. Docking of arabinofuranosyl-ferulate in the LbFAE structure highlights the restricted active site and lends further support to our hypothesis that LbFAE is specific for single FA side chains in arabinoxylan.

The impact of various β-glucans on the gut microbiome and immune system of vertebrates is becoming increasingly recognized. Besides the fundamental interest in understanding how β-glucans support human and animal health, enzymes that metabolize β-glucans are of interest for hemicellulose bioprocessing. Our earlier metagenomic analysis of the moose rumen microbiome identified a gene coding for a bacterial enzyme with a possible role in β-glucan metabolization. Here, we report that the enzyme, mrbExg5, has exo-β-1,3-glucanase activity on β-1,3-linked glucooligosaccharides and laminarin, but not on β-1,6- or β-1,4-glycosidic bonds. Longer oligosaccharides are good substrates, while shorter substrates are readily transglycosylated into longer products. The enzyme belongs to glycoside hydrolase subfamily GH5_44, which is a close phylogenetic neighbor of the subfamily GH5_9 exo-β-1,3-glucanases of the yeasts Saccharomyces cerevisiae and Candida albicans. The crystal structure shows that unlike the eukaryotic relatives, mrbExg5 is a functional homodimer with a binding region characterized by: (i) subsite +1 can accommodate a branched sugar on the β-1,3-glucan backbone; (ii) subsite +2 is restricted to exclude backbone substituents; and (iii) a fourth subsite (+3) formed by a unique loop. mrbExg5 is the first GH5_44 enzyme to be structurally characterized, and the first bacterial GH5 with exo-β-1,3-glucanase activity.

The termite Reticulitermes flavipes causes extensive damage due to the high efficiency and broad specificity of the ligno- and hemicellulolytic enzyme systems produced by its symbionts. Thus, the R. flavipes gut microbiome is expected to constitute an excellent source of enzymes that can be used for the degradation and valorization of plant biomass. The symbiont Opitutaceae bacterium strain TAV5 belongs to the phylum Verrucomicrobia and thrives in the hindgut of R. flavipes. The sequence of the gene with the locus tag opit5_10225 in the Opitutaceae bacterium strain TAV5 genome has been classified as a member of glycoside hydrolase family 5 (GH5), and provisionally annotated as an endo-beta-mannanase. We characterized biochemically and structurally the opit5_10225 gene product, and show that the enzyme, Op5Man5, is an exo-beta-1,4-mannosidase [EC 3.2.1.25] that is highly specific for beta-1,4-mannosidic bonds in mannooligosaccharides and ivory nut mannan. The structure of Op5Man5 was phased using electron cryo-microscopy and further determined and refined at 2.2 angstrom resolution using X-ray crystallography. Op5Man5 features a 200-kDa large homotrimer composed of three modular monomers. Despite insignificant sequence similarity, the structure of the monomer, and homotrimeric assembly are similar to that of the GH42-family beta-galactosidases and the GH164-family exo-beta-1,4-mannosidase Bs164 from Bacteroides salyersiae. To the best of our knowledge Op5Man5 is the first structure of a glycoside hydrolase from a bacterial symbiont isolated from the R. flavipes digestive tract, as well as the first example of a GH5 glycoside hydrolase with a GH42 beta-galactosidase-type homotrimeric structure.

Protein glycosylation constitutes a critical post-translational modification that supports a vast number of biological functions in living organisms across all domains of life. A seemingly boundless number of enzymes, glycosyltransferases, are involved in the biosynthesis of these protein-linked glycans. Few glycanbiosynthetic glycosyltransferases have been characterized in vitro, mainly due to the majority being integral membrane proteins and the paucity of relevant acceptor substrates. The crenarchaeote Pyrobaculum calidifontis belongs to the TACK superphylum of archaea (Thaumarchaeota, Aigarchaeota, Crenarchaeota, Korarchaeota) that has been proposed as an eukaryotic ancestor. In archaea, N-glycans are mainly found on cell envelope surface-layer proteins, archaeal flagellins and pili. Archaeal N-glycans are distinct from those of eukaryotes, but one noteworthy exception is the high-mannose N-glycan produced by P. calidifontis, which is similar in sugar composition to the eukaryotic counterpart. Here, we present the characterization and crystal structure of the first member of a crenarchaeal membrane glycosyltransferase, PcManGT. We show that the enzyme is a GDP-, dolichylphosphate-, and manganese-dependent mannosyltransferase. The membrane domain of PcManGT includes three transmembrane helices that topologically coincide with "half' of the sixtransmembrane helix cellulose-binding tunnel in Rhodobacter spheroides cellulose synthase BcsA. Conceivably, this "half tunnel" would be suitable for binding the dolichylphosphate-linked acceptor substrate. The PcManGT gene (Pcal_0472) is located in a large gene cluster comprising 14 genes of which 6 genes code for glycosyltransferases, and we hypothesize that this cluster may constitute a crenarchaeal N-glycosylation (PNG) gene cluster.

Carbohydrates are a primary energy source for all living organisms, but importantly, they also participate in a number of life-sustaining biological processes, e.g. cell signaling and cell-wall synthesis. The first part of the thesis examines glycosyltransferases that play a crucial role in the biosynthesis of N-glycans. Precursors to eukaryotic N-glycans are synthesized in the endoplasmic reticulum (ER) in the form of a lipid-bound oligosaccharide, which is then transferred to a nascent protein. The first seven sugar units are assembled on the cytoplasmic side of the ER, which is performed by glycosyltransferases that use nucleotide sugars as donors. The mannosyl transferase PcManGT is produced by the archaeon Pyrobaculum calidifontis, and the biochemical and structural results presented in the thesis suggest that the enzyme may be a counterpart to the glycosyltransferase Alg1 that participates in the biosynthesis of N-glycans in eukaryotes. Within the ER (in the lumen), activated dolichol-bound sugars are used as donor substrates instead of nucleotide sugars for glycosyltransferases that synthesize N-glycans. The glycosyltransferase dolichylphosphate mannose synthase (DPMS) catalyzes the formation of dolichylphosphate mannose, which is one of these dolichyl-bound sugars. The structure and function were studied for DPMS from Pyrococcus furiosus using protein X-ray crystallographic and biochemical methods and a new assay based on proteoliposomes was designed. The second part of the thesis focuses on glycoside hydrolases from bacteria that break down oligo- and polysaccharides. In one of the studies, a bacterial glycoside hydrolase from the acne bacterium Cutibacterium acnes was characterized. The enzyme was shown to be able to break down the host's N-glycans, which can be used as nutrients or perhaps even evade detection of the immune system. This study also suggests a cytoplasmic biosynthetic pathway for the formation of N-glycans in the acne bacterium. In another study, a glycoside hydrolase from a bacterium living in the moose rumen was characterized. The enzyme was shown to be able to break down β-1,3-glucans, which is a property that can be used industrially for biomass treatment.

Kolhydrater utgör en primär energikälla för alla levande organismer, man deltar dessutom i mängd livsuppehållande biologiska processer, t.ex. cellsignalering och cellväggssyntes. Den första delen av avhandlingen undersöker glykosyltransferaser som spelar en avgörande roll för biosyntes av N-glykaner. Förstadier till eukaryota N-glykaner syntetiseras i det endoplasmatiska nätverket (ER) i form av en lipidbunden oligosackarid som sedan överförs till ett nybildat protein. De första sju sockerenheterna sätts ihop på den cytoplasmatiska sidan av ER, vilket sker med hjälp av glykosyltransferaser som använder nukleotidsockerföreningar som kan donera sockerenheter. Mannosyltransferaset PcManGT produceras av arkebakterien Pyrobaculum calidifontis och de biokemiska och strukturella resultat som presenteras i avhandlingen tyder på att enzymet kan vara en motsvarighet till glykosyltransferaset Alg1 som deltar i biosyntes av N-glykaner i eukaryoter. Inuti ER (i lumen) används aktiverade dolikolbundna socker istället för nukleotidsockerföreningar som substrat för glykosyltransferaser som syntetiserar N-glykaner. Glykosyltransferaset dolikolfosfatmannossyntas (DPMS) katalyserar bildandet av dolikolfosfatmannos vilket är en av dessa dolikolbundna sockerföreningar. Struktur och funktion studerades för DPMS från Pyrococcus furiosus med hjälp av proteinröntgenkristallografi samt biokemiska metoder samt en ny analysmetid baserad på proteoliposomer. Den andra delen av avhandlingen fokuserar på glykosidhydrolaser från bakterier som bryter ned oligo- och polysackarider. I en av studierna karakteriserades ett bakteriellt glykosidhydrolas från aknebakterien Cutibacterium acnes. Enzymet visade sig kunna bryta ned värdorganismens N-glykaner vilka kan användas som näring eller kanske även i syfte att undgå upptäckt av immunförsvaret. Studien föreslår även en cytoplasmatisk biosyntesväg för bildande av N-glykaner i aknebakterien. I en annan studie karakteriserades ett glykosidhydrolas från en bakterie som lever i älgvåmmen. Enzymet visade sig kunna bryta ned β-1,3- glukaner vilket är en egenskap som kan användas för i industriella tillämpningar för bearbetning av biomassa.

Kohlenhydrate sind eine primäre Energiequelle für alle lebenden Organismen, aber vor allem sind sie an einer großen Anzahl von lebensnotwendigen biologischen Prozessen, z.B. Zellsignalwegen und Zellwandaufbau, beteiligt. Der erste Teil dieser Doktorarbeit untersucht Glycosyltransferasen, welche entscheidend für die Synthese von N-Glycanen sind. Die Vorläufer der eukaryotischen N-Glycane werden im endoplasmatischen Retikulum (ER) als Lipid-gebundene Oligosaccharide synthetisiert, bevor sie auf das entstehende Protein transferiert werden. Die ersten sieben Zucker werden auf der zytoplasmatischen Seite der ER-Membran von Glycosyltransferasen, welche Nukleotidzucker als Donor nutzen, transferiert. Die Mannosyltransferase PcManGT wird von den Archaea Pyrobaculum calidifontis produziert, und die in dieser Doktorarbeit präsentierten biochemischen und strukturellen Resultate deuten darauf hin, dass das Enzym ein Pedant zu dem eukaryotischen Enzym Alg1 ist, welches Teil der eukaryotischen N-Glycan Biosynthese ist. Im ER (ERlumen) werden aktivierte Dolichol-gebundene Zucker, statt Nukleotidzuckern als Donor, für N-Glycan synthetisierende Glycosyltransferasen bereitgestellt. Die Glycosyltransferase Dolichylphosphate Mannose Synthase (DPMS) katalysiert mit der Bildung von Dolichylphosphate Mannose, einen dieser Dolicholgebundene Zucker. Die Struktur und Funktion der DPMS von Pyrococcus furiosus wurde mit Hilfe von sowohl X-ray Kristallographie als auch biochemischen Methoden untersucht und ein neuer Assay basierend auf Proteoliposomen designt. Im zweiten Teil der Doktorarbeit fokussiert sich auf bakterielle Glycosidasen welche Oligo- und Polysaccharide abbauen. Charakterisiert wurde die bakterielle Glycosidase von dem Aknebakterium Cutibacterium acnes. Das Enzym kann Teile des Wirt N-Glycans abbauen, wodurch Nährstoffe bereitgestellt werden oder die Erkennung des Immunsystems vermieden wird. Zusätzlich wird auch ein potentieller N-Glycan Abbauweg des Aknebakterium vorgeschlagen. In einer weiteren Studie wurde eine Glucosidase von einem Elchmagenbakterium charakterisiert. Das Enzym war in der Lage β-1,3-Glucane abzubauen, welches Anwendung in der industriellen Biomassebehandlung finden könnte.

While in search of an enzyme for the conversion of xylose to xylitol at elevated temperatures, a xylose reductase (XR) gene was identified in the genome of the thermophilic fungus Chaetomium thermophilum. The gene was heterologously expressed in Escherichia coli as a His6-tagged fusion protein and characterized for function and structure. The enzyme exhibits dual cofactor specificity for NADPH and NADH and prefers D-xylose over other pentoses and investigated hexoses. A homology model based on a XR from Candida tenuis was generated and the architecture of the cofactor binding site was investigated in detail. Despite the outstanding thermophilicity of its host the enzyme is, however, not thermostable.

Commensal and pathogenic bacteria have evolved efficient enzymatic pathways to feed on host carbohydrates, including protein-linked glycans. Most proteins of the human innate and adaptive immune system are glycoproteins where the glycan is critical for structural and functional integrity. Besides enabling nutrition, the degradation of host N-glycans serves as a means for bacteria to modulate the host's immune system by for instance removing N-glycans on immunoglobulin G. The commensal bacterium Cutibacterium acnes is a gram-positive natural bacterial species of the human skin microbiota. Under certain circumstances, C. acnes can cause pathogenic conditions, acne vulgaris, which typically affects 80% of adolescents, and can become critical for immunosuppressed transplant patients. Others have shown that C. acnes can degrade certain host O-glycans, however, no degradation pathway for host N-glycans has been proposed. To investigate this, we scanned the C. acnes genome and were able to identify a set of gene candidates consistent with a cytoplasmic N-glycan-degradation pathway of the canonical eukaryotic N-glycan core. We also found additional gene sequences containing secretion signals that are possible candidates for initial trimming on the extracellular side. Furthermore, one of the identified gene products of the cytoplasmic pathway, AEE72695, was produced and characterized, and found to be a functional, dimeric exo-β-1,4-mannosidase with activity on the β-1,4 glycosidic bond between the second N-acetylglucosamine and the first mannose residue in the canonical eukaryotic N-glycan core. These findings corroborate our model of the cytoplasmic part of a C. acnes N-glycan degradation pathway.

The catalytic activity of the allosteric enzyme pyruvate decarboxylase from yeast is strictly controlled by its own substrate pyruvate via covalent binding at a separate regulatory site. Kinetic studies, chemical modifications, cross-linking, small-angle X-ray scattering, and crystal structure analyses have led to a detailed understanding of the substrate activation mechanism at an atomic level with C221 as the core moiety of the regulatory site. To characterize the individual role of the residues adjacent to C221, we generated variants H92F, H225F, H310F, A287G, S311A, and C221A/C222A. The integrity of the protein structure of the variants was established by small-angle X-ray scattering measurements. The analyses of both steady state and transient kinetic data allowed the identification of the individual roles of the exchanged side chains during allosteric enzyme activation. In each case, the kinetic pattern of activation was modulated but not completely abolished. Despite the crucial role of C221, the covalent binding of pyruvate is not obligate for enzyme activation but is a requirement for a kinetically efficient transition from the inactive to the active state. Moreover, only one of the three histidines guiding the activator molecule to the binding pocket, H310, specifically interacts with C221. H310 stabilizes the thiolate form of C221, ensuring a rapid nucleophilic attack of the thiolate sulfur on C2 of the regulatory pyruvate, thus forming a regulatory dyad. The influence of the other two histidines is less pronounced. Substrate activation is slightly weakened for A287G and significantly retarded for S311A.

Protein glycosylation is a critical protein modification. In biogenic membranes of eukaryotes and archaea, these reactions require activated mannose in the form of the lipid conjugate dolichylphosphate mannose (Dol-P-Man). The membrane protein dolichylphosphate mannose synthase (DPMS) catalyzes the reaction whereby mannose is transferred from GDP-mannose to the dolichol carrier Dol-P, to yield Dol-P-Man. Failure to produce or utilize Dol-P-Man compromises organism viability, and in humans, several mutations in the human dpm1 gene lead to congenital disorders of glycosylation (CDG). Here, we report three high-resolution crystal structures of archaeal DPMS from Pyrococcus furiosus, in complex with nucleotide, donor, and glycolipid product. The structures offer snapshots along the catalytic cycle, and reveal how lipid binding couples to movements of interface helices, metal binding, and acceptor loop dynamics to control critical events leading to Dol-P-Man synthesis. The structures also rationalize the loss of dolichylphosphate mannose synthase function in dpm1-associated CDG.The generation of glycolipid dolichylphosphate mannose (Dol-P-Man) is a critical step for protein glycosylation and GPI anchor synthesis. Here the authors report the structure of dolichylphosphate mannose synthase in complex with bound nucleotide and donor to provide insight into the mechanism of Dol-P-Man synthesis.