Agar degradation by microorganisms and agar-degrading enzymes

Agar is a mixture of heterogeneous galactans, mainly composed of 3,6-anhydro-l-galactoses (or l-galactose-6-sulfates) d-galactoses and l-galactoses (routinely in the forms of 3,6-anhydro-l-galactoses or l-galactose-6-sulfates) alternately linked by β-(1,4) and α-(1,3) linkages. It is a major component of the cell walls of red algae and has been used in a variety of laboratory and industrial applications, owing to its jellifying properties. Many microorganisms that can hydrolyze and metabolize agar as a carbon and energy source have been identified in seawater and marine sediments. Agarolytic microorganisms commonly produce agarases, which catalyze the hydrolysis of agar. Numerous agarases have been identified in microorganisms of various genera. They are classified according to their cleavage pattern into three types—α-agarase, β-agarase, and β-porphyranase. Although, in a broad sense, many other agarases are involved in complete hydrolysis of agar, most of those identified are β-agarases. In this article we review agarolytic microorganisms and their agar-hydrolyzing systems, covering β-agarases as well as α-agarases, α-neoagarobiose hydrolases, and β-porphyranases, with emphasis on the recent discoveries. We also present an overview of the biochemical and structural characteristics of the various types of agarases. Further, we summarize and compare the agar-hydrolyzing systems of two specific microorganisms: Gram-negative Saccharophagus degradans 2–40 and Gram-positive Streptomyces coelicolor A3(2). We conclude with a brief discussion of the importance of agarases and their possible future application in producing oligosaccharides with various nutraceutical activities and in sustainably generating stock chemicals for biorefinement and bioenergy.     

Comparative Characterization of Two Marine Alginate Lyases from Zobellia galactanivorans Reveals Distinct Modes of Action and Exquisite Adaptation to Their Natural Substrate

Cell walls of brown algae are complex supramolecular assemblies containing various original, sulfated, and carboxylated polysaccharides. Among these, the major marine polysaccharide component, alginate, represents an important biomass that is successfully turned over by the heterotrophic marine bacteria. In the marine flavobacterium Zobellia galactanivorans, the catabolism and uptake of alginate are encoded by operon structures that resemble the typical Bacteroidetes polysaccharide utilization locus. The genome of Z. galactanivorans contains seven putative alginate lyase genes, five of which are localized within two clusters comprising additional carbohydrate-related genes. This study reports on the detailed biochemical and structural characterization of two of these. We demonstrate here that AlyA1_PL7 is an endolytic guluronate lyase, and AlyA5 cleaves unsaturated units, α-l-guluronate or β-d-manuronate residues, at the nonreducing end of oligo-alginates in an exolytic fashion. Despite a common jelly roll-fold, these striking differences of the mode of action are explained by a distinct active site topology, an open cleft in AlyA1_PL7, whereas AlyA5 displays a pocket topology due to the presence of additional loops partially obstructing the catalytic groove. Finally, in contrast to PL7 alginate lyases from terrestrial bacteria, both enzymes proceed according to a calcium-dependent mechanism suggesting an exquisite adaptation to their natural substrate in the context of brown algal cell walls.     

Structure and function of an arabinoxylan-specific xylanase

The enzymatic degradation of plant cell walls plays a central role in the carbon cycle and is of increasing environmental and industrial significance. The enzymes that catalyze this process include xylanases that degrade xylan, a β-1,4-xylose polymer that is decorated with various sugars. Although xylanases efficiently hydrolyze unsubstituted xylans, these enzymes are unable to access highly decorated forms of the polysaccharide, such as arabinoxylans that contain arabinofuranose decorations. Here, we show that a Clostridium thermocellum enzyme, designated CtXyl5A, hydrolyzes arabinoxylans but does not attack unsubstituted xylans. Analysis of the reaction products generated by CtXyl5A showed that all the oligosaccharides contain an O3 arabinose linked to the reducing end xylose. The crystal structure of the catalytic module (CtGH5) of CtXyl5A, appended to a family 6 noncatalytic carbohydrate-binding module (CtCBM6), showed that CtGH5 displays a canonical (α/β)(8)-barrel fold with the substrate binding cleft running along the surface of the protein. The catalytic apparatus is housed in the center of the cleft. Adjacent to the -1 subsite is a pocket that could accommodate an l-arabinofuranose-linked α-1,3 to the active site xylose, which is likely to function as a key specificity determinant. CtCBM6, which adopts a β-sandwich fold, recognizes the termini of xylo- and gluco-configured oligosaccharides, consistent with the pocket topology displayed by the ligand-binding site. In contrast to typical modular glycoside hydrolases, there is an extensive hydrophobic interface between CtGH5 and CtCBM6, and thus the two modules cannot function as independent entities.     

Crystal structure of a key enzyme in the agarolytic pathway, α-neoagarobiose hydrolase from Saccharophagus degradans 2-40

In agarolytic microorganisms, α-neoagarobiose hydrolase (NABH) is an essential enzyme to metabolize agar because it converts α-neoagarobiose (O-3,6-anhydro-alpha-l-galactopyranosyl-(1,3)-d-galactose) into fermentable monosaccharides (d-galactose and 3,6-anhydro-l-galactose) in the agarolytic pathway. NABH can be divided into two biological classes by its cellular location. Here, we describe a structure and function of cytosolic NABH from Saccharophagus degradans 2–40 in a native protein and d-galactose complex determined at 2.0 and 1.55 Å, respectively. The overall fold is organized in an N-terminal helical extension and a C-terminal five-bladed β-propeller catalytic domain. The structure of the enzyme–ligand (d-galactose) complex predicts a +1 subsite in the substrate binding pocket. The structural features may provide insights for the evolution and classification of NABH in agarolytic pathways.     

Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum

Inverting cellobiose phosphorylase (CtCBP) and cellodextrin phosphorylase (CtCDP) from Clostridium thermocellum ATCC27405 of glycoside hydrolase family 94 catalysed reverse phosphorolysis to produce cellobiose and cellodextrins in 57% and 48% yield from α-d-glucose 1-phosphate as donor with glucose and cellobiose as acceptor, respectively. Use of α-d-glucosyl 1-fluoride as donor increased product yields to 98% for CtCBP and 68% for CtCDP. CtCBP showed broad acceptor specificity forming β-glucosyl disaccharides with β-(1→4)- regioselectivity from five monosaccharides as well as branched β-glucosyl trisaccharides with β-(1→4)-regioselectivity from three (1→6)-linked disaccharides. CtCDP showed strict β-(1→4)-regioselectivity and catalysed linear chain extension of the three β-linked glucosyl disaccharides, cellobiose, sophorose, and laminaribiose, whereas 12 tested monosaccharides were not acceptors. Structure analysis by NMR and ESI-MS confirmed two β-glucosyl oligosaccharide product series to represent novel compounds, i.e. β-d-glucopyranosyl-[(1→4)-β-d-glucopyranosyl]_n-(1→2)-d-glucopyranose, and β-d-glucopyranosyl-[(1→4)-β-d-glucopyranosyl]_n-(1→3)-d-glucopyranose (n = 1–7). Multiple sequence alignment together with a modelled CtCBP structure, obtained using the crystal structure of Cellvibrio gilvus CBP in complex with glucose as a template, indicated differences in the subsite +1 region that elicit the distinct acceptor specificities of CtCBP and CtCDP. Thus Glu636 of CtCBP recognized the C1 hydroxyl of β-glucose at subsite +1, while in CtCDP the presence of Ala800 conferred more space, which allowed accommodation of C1 substituted disaccharide acceptors at the corresponding subsites +1 and +2. Furthermore, CtCBP has a short Glu496-Thr500 loop that permitted the C6 hydroxyl of glucose at subsite +1 to be exposed to solvent, whereas the corresponding longer loop Thr637–Lys648 in CtCDP blocks binding of C6-linked disaccharides as acceptors at subsite +1. High yields in chemoenzymatic synthesis, a novel regioselectivity, and novel oligosaccharides including products of CtCDP catalysed oligosaccharide oligomerisation using α-d-glucosyl 1-fluoride, all together contribute to the formation of an excellent basis for rational engineering of CBP and CDP to produce desired oligosaccharides.     

Structure and activity of Paenibacillus polymyxa xyloglucanase from glycoside hydrolase family 44

The enzymatic degradation of plant polysaccharides is emerging as one of the key environmental goals of the early 21st century, impacting on many processes in the textile and detergent industries as well as biomass conversion to biofuels. One of the well known problems with the use of nonstarch (nonfood)-based substrates such as the plant cell wall is that the cellulose fibers are embedded in a network of diverse polysaccharides, including xyloglucan, that renders access difficult. There is therefore increasing interest in the "accessory enzymes," including xyloglucanases, that may aid biomass degradation through removal of "hemicellulose" polysaccharides. Here, we report the biochemical characterization of the endo-β-1,4-(xylo)glucan hydrolase from Paenibacillus polymyxa with polymeric, oligomeric, and defined chromogenic aryl-oligosaccharide substrates. The enzyme displays an unusual specificity on defined xyloglucan oligosaccharides, cleaving the XXXG-XXXG repeat into XXX and GXXXG. Kinetic analysis on defined oligosaccharides and on aryl-glycosides suggests that both the -4 and +1 subsites show discrimination against xylose-appended glucosides. The three-dimensional structures of PpXG44 have been solved both in apo-form and as a series of ligand complexes that map the -3 to -1 and +1 to +5 subsites of the extended ligand binding cleft. Complex structures are consistent with partial intolerance of xylosides in the -4' subsites. The atypical specificity of PpXG44 may thus find use in industrial processes involving xyloglucan degradation, such as biomass conversion, or in the emerging exciting applications of defined xyloglucans in food, pharmaceuticals, and cellulose fiber modification.     

Sandalwood fragrance biosynthesis involves sesquiterpene synthases of both the terpene synthase (TPS)-a and TPS-b subfamilies, including santalene synthases

Sandalwood oil is one of the worlds most highly prized fragrances. To identify the genes and encoded enzymes responsible for santalene biosynthesis, we cloned and characterized three orthologous terpene synthase (TPS) genes SaSSy, SauSSy, and SspiSSy from three divergent sandalwood species; Santalum album, S. austrocaledonicum, and S. spicatum, respectively. The encoded enzymes catalyze the formation of α-, β-, epi-β-santalene, and α-exo-bergamotene from (E,E)-farnesyl diphosphate (E,E-FPP). Recombinant SaSSy was additionally tested with (Z,Z)-farnesyl diphosphate (Z,Z-FPP) and remarkably, found to produce a mixture of α-endo-bergamotene, α-santalene, (Z)-β-farnesene, epi-β-santalene, and β-santalene. Additional cDNAs that encode bisabolene/bisabolol synthases were also cloned and functionally characterized from these three species. Both the santalene synthases and the bisabolene/bisabolol synthases reside in the TPS-b phylogenetic clade, which is more commonly associated with angiosperm monoterpene synthases. An orthologous set of TPS-a synthases responsible for formation of macrocyclic and bicyclic sesquiterpenes were characterized. Strict functionality and limited sequence divergence in the santalene and bisabolene synthases are in contrast to the TPS-a synthases, suggesting these compounds have played a significant role in the evolution of the Santalum genus.     

一株多糖降解菌的分离、鉴定与琼脂糖降解能力

【目的】筛选海洋来源的多糖降解菌,分析其多糖降解能力并初探机制。【方法】碘液染色法从海泥中初筛琼脂糖降解菌,唯一碳源生长法分析菌株的多糖利用能力,克隆16S rRNA基因以分析系统分类地位。用硫酸铵沉淀法制备胞外粗酶制剂,DNS-还原糖法测定琼胶酶活性,活性染色法分析胞外琼胶酶系的组成特征。分离、纯化琼脂糖的酶解产物,通过TLC测定寡糖Rf值、阳离子质谱测定分子量。【结果】分离到1株能液化琼脂糖的海洋细菌JZB09,鉴定至桃色杆菌属(Persicobacter)。JZB09能利用11种不同的多糖为唯一碳源生长,在利用琼脂糖、纤维素和木聚糖时生长较好。胞外粗酶制剂的琼胶酶活力约77.2U/mg,含有至少2条琼胶酶,大小约45kDa、70kDa。酶制剂降解琼脂糖后的产物是系列新琼寡糖,四糖是主产物,表明β-琼胶酶在胞外琼胶酶系降解琼脂糖时起关键作用。【结论】海洋细菌Persicobacter sp.JZB09是1株多能型多糖降解菌,可分泌β-琼胶酶降解琼脂糖且活性显著,具有潜在开发价值。     

Tyrosine 105 and threonine 212 at outermost substrate binding subsites -6 and +4 control substrate specificity, oligosaccharide cleavage patterns, and multiple binding modes of barley alpha-amylase 1

The role in activity of outer regions in the substrate binding cleft in alpha-amylases is illustrated by mutational analysis of Tyr(105) and Thr(212) localized at subsites -6 and +4 (substrate cleavage occurs between subsites -1 and +1) in barley alpha-amylase 1 (AMY1). Tyr(105) is conserved in plant alpha-amylases whereas Thr(212) varies in these and related enzymes. Compared with wild-type AMY1, the subsite -6 mutant Y105A has 140, 15, and <1% activity (k(cat)/K(m)) on starch, amylose DP17, and 2-chloro-4-nitrophenyl beta-d-maltoheptaoside, whereas T212Y at subsite +4 has 32, 370, and 90% activity, respectively. Thus engineering of aromatic stacking interactions at the ends of the 10-subsite long binding cleft affects activity very differently, dependent on the substrate. Y105A dominates in dual subsite -6/+4 [Y105A/T212(Y/W)]AMY1 mutants having almost retained and low activity on starch and oligosaccharides, respectively. Bond cleavage analysis of oligosaccharide degradation by wild-type and mutant AMY1 supports that Tyr(105) is critical for binding at subsite -6. Substrate binding is improved by T212(Y/W) introduced at subsite +4 and the [Y105A/T212(Y/W)]AMY1 double mutants synergistically enhanced productive binding of the substrate aglycone. The enzymatic properties of the series of AMY1 mutants suggest that longer substrates adopt several binding modes. This is in excellent agreement with computed distinct multiple docking solutions observed for maltododecaose at outer binding areas of AMY1 beyond subsites -3 and +3.     

Structural and kinetic insights reveal that the amino acid pair Gln-228/Asn-254 modulates the transfructosylating specificity of Schwanniomyces occidentalis β-fructofuranosidase, an enzyme that produces prebiotics

Schwanniomyces occidentalis β-fructofuranosidase (Ffase) is a GH32 dimeric enzyme that releases fructose from the nonreducing end of various oligosaccharides and essential storage fructans such as inulin. It also catalyzes the transfer of a fructosyl unit to an acceptor producing 6-kestose and 1-kestose, prebiotics that stimulate the growth of bacteria beneficial for human health. We report here the crystal structure of inactivated Ffase complexed with fructosylnystose and inulin, which shows the intricate net of interactions keeping the substrate tightly bound at the active site. Up to five subsites were observed, the sugar unit located at subsite +3 being recognized by interaction with the β-sandwich domain of the adjacent subunit within the dimer. This explains the high activity observed against long substrates, giving the first experimental evidence of the direct role of a GH32 β-sandwich domain in substrate binding. Crucial residues were mutated and their hydrolase/transferase (H/T) activities were fully characterized, showing the involvement of the Gln-228/Asn-254 pair in modulating the H/T ratio and the type β(2-1)/β(2-6) linkage formation. We generated Ffase mutants with new transferase activity; among them, Q228V gives almost specifically 6-kestose, whereas N254T produces a broader spectrum product including also neokestose. A model for the mechanism of the Ffase transfructosylation reaction is proposed. The results contribute to an understanding of the molecular basis regulating specificity among GH-J clan members, which represent an interesting target for rational design of enzymes, showing redesigned activities to produce tailor-made fructooligosaccharides.     

Kinetics and stereochemistry of the Cellulomonas fimi β-mannanase studied using 1H-NMR

Endo–1,4-β-mannanases (β-mannanases) randomly hydrolyse the mannosidic bonds within the main chain of various mannans and heteromannans. Some of these polysaccharides are hemicelluloses, a major part of the plant cell-wall. The β-mannanases have been assigned to family 5 and 26 of the glycoside hydrolase clan A. This work presents a detailed kinetic analysis of the family 26 β-mannanase CfMan26A from the soil-bacterium Cellulomonas fimi. The full-length enzyme consists of five modules: a family 26 catalytic module, an immunoglobulin-like module, a mannan-binding module, a surface layer homology-module and a module of unknown function. A truncated variant consisting of the catalytic module and the immunoglobulin-like module was used in these studies. The degradation of mannotriose, mannotetraose and mannopentaose was studied by ¹H-NMR. First, the mutarotation of one of the hydrolysis products (mannose) was determined to be 1.7 10^?5s^?1 at 5°C and pH 5.0. As expected for a family 26 glycoside hydrolase, the hydrolysis was shown to proceed with overall retention of the anomeric configuration. Many ‘retaining’ enzymes can perform transglycosylation reactions. However, no transglycosylation could be detected. Kinetic constants were calculated from progress curves using computer simulation. It was revealed that the ?3 subsite had a greater impact on the apparent k_cat/K_m ratio (the catalytic efficiency) than the +2 subsite. The β-anomer of mannotriose was hydrolysed 1000-times more efficiently than the α-anomer indicating selectivity for the β- over the α-anomer in the +1 subsite. With background information from the previous published 3D-structure of the truncated variant of Man26A, a structural explanation for the observations is discussed.     

NMR study on hydroxy protons of κ- and κ/μ-hybrid carrageenan oligosaccharides: experimental evidence of hydrogen bonding and chemical exchange interactions in κ/μ oligosaccharides

The hydroxy protons of κ- and κ/μ-hybrid carrageenan oligosaccharides have been studied by NMR spectroscopy in 85% H(2)O/15% acetone-d(6). Hydration and hydrogen bonding interactions in di- (κ), tetra- (κκ), hexa (κκκ), and octa- (κκκκ) κ-oligosaccharides and hexa- (κμκ), octa- (κμμκ), and deca- (κμμμκ) κ/μ-oligosaccharides have been investigated by measuring the chemical shifts, temperature coefficients, and chemical exchange of the hydroxy protons. These NMR parameters indicate that no strong and persistent intramolecular hydrogen bonds involving hydroxy protons stabilize the structure of κ-carrageenan oligosaccharides in aqueous solution. In the κ/μ-oligosaccharides, the presence of chemical exchange between OH3 of α-d-Gal-6-sulfate (D6S) and OH2 of β-d-Gal-4-sulfate (G4S) across the β-d-Gal-4-S-(1→4)-α-d-Gal-6-S linkage reveals the existence of a weak hydrogen bond interaction between the two hydroxyl groups. The smaller temperature coefficients of OH2_D6S and OH3_D6S indicate reduced hydration, interpreted as spatial proximity to the 4-sulfate group and O5 ring oxygen of the neighboring G4S residues, respectively. These first experimental results on the conformation of κ/μ-carrageenan oligosaccharides shine light on the potential role of "kinks" in the properties of the three-dimensional carrageenan gel network.     

Sequence and Structural Features of Subsite Residues in GH10 and GH11 Xylanases

Xylanases are the enzymes that breakdown complex plant cell wall polysaccharide xylan into xylose by hydrolysing the β-(1→4) glycosidic linkage between xylosides. They mainly belong to the families GH10 and GH11 of the glycoside hydrolase claβs of enzymes. GH10 xylanases have (α/β)₈-barrel type of fold whereas GH11 xylanases have β-jelly roll type of fold. Both enzymes have several substrate binding subsites. This study analysed in detail the sequence and structural conservation of subsites residues by examining their 3D structures crystallized with homoxylan or its non-hydrolysable form as substrate. A total of 19 structures from GH10 and 6 structures from GH11 were analysed. It was found that in GH10 the subsites -3 to -1 consisted of conserved residues, whereas in GH11 subsites -1, -3 and +1 were found to be conserved. The substrate and subsite interaction analysed based on the presence of h-bonds and CH-π interactions showed that Face-to-Face or Edge-to-Face CH-π interactions are formed in the subsites of GH10, whereas such specific CH-π interactions were no at all observed in case of GH11 xylanases. The spatial conservation of subsite residues was also analysed using a distance matrix based approach. It was found that in GH10 xylanases conserved residues have conserved spatial position of those residues as opposed to GH11 enzymes where in subsites -2 and +2 conserved residues showed non-conservation in their spatial positions. The results presented in this study can be used in discovering new xylanases and in the engineering highly efficient xylanases.     

Functional analysis of hyperthermophilic endocellulase from Pyrococcus horikoshii by crystallographic snapshots

A hyperthermophilic membrane-related β-1,4-endoglucanase (family 5, cellulase) of the archaeon Pyrococcus horikoshii was found to be capable of hydrolysing cellulose at high temperatures. The hyperthermophilic cellulase has promise for applications in biomass utilization. To clarify its detailed function, we determined the crystal structures of mutants of the enzyme in complex with either the substrate or product ligands. We were able to resolve different kinds of complex structures at 1.65-2.01?? (1??=0.1?nm). The structural analysis of various mutant enzymes yielded a sequence of crystallographic snapshots, which could be used to explain the catalytic process of the enzyme. The substrate position is fixed by the alignment of one cellobiose unit between the two aromatic amino acid residues at subsites +1 and +2. During the enzyme reaction, the glucose structure of cellulose substrates is distorted at subsite -1, and the β-1,4-glucoside bond between glucose moieties is twisted between subsites -1 and +1. Subsite -2 specifically recognizes the glucose residue, but recognition by subsites +1 and +2 is loose during the enzyme reaction. This type of recognition is important for creation of the distorted boat form of the substrate at subsite -1. A rare enzyme-substrate complex was observed within the low-activity mutant Y299F, which suggested the existence of a trapped ligand structure before the formation by covalent bonding of the proposed intermediate structure. Analysis of the enzyme-substrate structure suggested that an incoming water molecule, essential for hydrolysis during the retention process, might be introduced to the cleavage position after the cellobiose product at subsites +1 and +2 was released from the active site.     

Ion-spray mass spectrometric analysis of glycosaminoglycan oligosaccharides

Oligosaccharides from hyaluronic acid and chondroitin 6-sulfate were prepared by digestion with testicular hyaluronidase and separated according to their degree of polymerization by gel-permeation chromatography. These materials were successively analyzed by negative-mode ion-spray mass spectrometry with an atmospheric-pressure ion source. An ion-spray interface was used to produce ions via the ion evaporation process, producing mass spectra containing a series of molecular species carrying multiple charges. Using two adjacent multiply charged molecular ions, the exact molecular weights up to the tetradecasaccharide were calculated with a precision of ±1 dalton. This type of mass spectrometry was also demonstrated to be feasible for the analysis of mixtures of oligosaccharides, including tetra-, hexa-, octa- and decasaccharides, from hyaluronic acid or chondroitin 6-sulfate without separation. Ion-spray mass spectrometry was thus shown to be applicable to the structural analysis of oligosaccharides from glycosaminoglycans.Abbreviations HA hyaluronic acid - Ch6S chondroitin 6-sulfate - GAG glycosaminoglycan - GlcA d-glucuronic acid - GlcNAc 2-acetamido-2-deoxy-d-glucose - GalNAc 2-acetamido-2-deoxy-d-galactose.     

A β1‐6/β1‐3 galactosidase from Bifidobacterium animalis subsp. lactis Bl‐04 gives insight into sub‐specificities of β‐galactoside catabolism within Bifidobacterium

The Bifidobacterium genus harbours several health promoting members of the gut microbiota. Bifidobacteria display metabolic specialization by preferentially utilizing dietary or host‐derived β‐galactosides. This study investigates the biochemistry and structure of a glycoside hydrolase family 42 (GH42) β‐galactosidase from the probiotic Bifidobacterium animalis subsp. lactis Bl‐04 (BlGal42A). BlGal42A displays a preference for undecorated β1‐6 and β1‐3 linked galactosides and populates a phylogenetic cluster with close bifidobacterial homologues implicated in the utilization of N‐acetyl substituted β1‐3 galactosides from human milk and mucin. A long loop containing an invariant tryptophan in GH42, proposed to bind substrate at subsite + 1, is identified here as specificity signature within this clade of bifidobacterial enzymes. Galactose binding at the subsite ? 1 of the active site induced conformational changes resulting in an extra polar interaction and the ordering of a flexible loop that narrows the active site. The amino acid sequence of this loop provides an additional specificity signature within this GH42 clade. The phylogenetic relatedness of enzymes targeting β1‐6 and β1‐3 galactosides likely reflects structural differences between these substrates and β1‐4 galactosides, containing an axial galactosidic bond. These data advance our molecular understanding of the evolution of sub‐specificities that support metabolic specialization in the gut niche.     

Structural and functional basis for substrate specificity and catalysis of levan fructotransferase

Levan is β-2,6-linked polymeric fructose and serves as reserve carbohydrate in some plants and microorganisms. Mobilization of fructose is usually mediated by enzymes such as glycoside hydrolase (GH), typically releasing a monosaccharide as a product. The enzyme levan fructotransferase (LFTase) of the GH32 family catalyzes an intramolecular fructosyl transfer reaction and results in production of cyclic difructose dianhydride, thus exhibiting a novel substrate specificity. The mechanism by which LFTase carries out these functions via the structural fold conserved in the GH32 family is unknown. Here, we report the crystal structure of LFTase from Arthrobacter ureafaciens in apo form, as well as in complexes with sucrose and levanbiose, a difructosacchride with a β-2,6-glycosidic linkage. Despite the similarity of its two-domain structure to members of the GH32 family, LFTase contains an active site that accommodates a difructosaccharide using the -1 and -2 subsites. This feature is unique among GH32 proteins and is facilitated by small side chain residues in the loop region of a catalytic β-propeller N-domain, which is conserved in the LFTase family. An additional oligosaccharide-binding site was also characterized in the β-sandwich C-domain, supporting its role in carbohydrate recognition. Together with functional analysis, our data provide a molecular basis for the catalytic mechanism of LFTase and suggest functional variations from other GH32 family proteins, notwithstanding the conserved structural elements.     

The role of subsite +2 of the Trichoderma reesei β-mannanase TrMan5A in hydrolysis and transglycosylation

The N-terminal catalytic module of β-mannanase TrMan5A from the filamentous fungus Trichoderma reesei is classified into family 5 of glycoside hydrolases. It is further classified in clan A with a (β/α)₈ barrel configuration and has two catalytic glutamates (E169 and E276). It has at least five other residues conserved in family 5. Sequence alignment revealed that an arginine (R171 in TrMan5A) is semi-conserved among β-mannanases in family 5. In a previously published mannobiose complex structure, this residue is positioned in hydrogen bonding distance from the C2 hydroxyl group of the mannose residue bound at the +2 subsite. To study the function of R171, mutants of this residue were constructed. The results show that arginine 171 is important for substrate binding and transglycosylation. A mutant of TrMan5A with the substitution R171K displayed retained activity on polymeric galactomannan but reduced activity on oligosaccharides due to an increase of K_m. While the wild-type enzyme produces mannobiose as dominant product from mannotetraose the R171K mutant shows an altered product profile, producing mannotriose and mannose. The cleavage pattern of mannotetraose was analysed with a method using isotope labelled water (H₂¹⁸O) and mass spectrometry which showed that the preferred productive binding mode of mannotetraose was shifted from subsite ?2 to +2 in the wild-type to subsite ?3 to +1 in the R171K mutant. Significant differences in product formation after manno-oligosaccharide incubation showed that the wild-type enzyme can perform transglycosylation on to saccharide acceptors while the R171K mutant cannot, likely due to loss of acceptor affinity. Interestingly, both enzymes show the ability to perform alcoholysis reactions with methanol and butanol, forming new β-linked glyco-conjugates. Furthermore, it appears that the wild-type enzyme produces mainly mannobiose conjugates using M₄ as substrate, while in contrast the R171K mutant produces mainly mannotriose conjugates, due to the altered subsite binding.     

Effects of essential carbohydrate/aromatic stacking interaction with Tyr100 and Phe259 on substrate binding of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp. 1011

The stacking interaction between a tyrosine residue and the sugar ring at the catalytic subsite -1 is strictly conserved in the glycoside hydrolase family 13 enzymes. Replacing Tyr100 with leucine in cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. 1011 to prevent stacking significantly decreased all CGTase activities. The adjacent stacking interaction with both Phe183 and Phe259 onto the sugar ring at subsite +2 is essentially conserved among CGTases. F183L/F259L mutant CGTase affects donor substrate binding and/or acceptor binding during transglycosylation [Nakamura et al. (1994) Biochemistry 33, 9929-9936]. To elucidate the precise role of carbohydrate/aromatic stacking interaction at subsites -1 and +2 on the substrate binding of CGTases, we analyzed the X-ray structures of wild-type (2.0 A resolution), and Y100L (2.2 A resolution) and F183L/F259L mutant (1.9 A resolution) CGTases complexed with the inhibitor, acarbose. The refined structures revealed that acarbose molecules bound to the Y100L mutant moved from the active center toward the side chain of Tyr195, and the hydrogen bonding and hydrophobic interaction between acarbose and subsites significantly diminished. The position of pseudo-tetrasaccharide binding in the F183L/F259L mutant was closer to the non-reducing end, and the torsion angles of glycosidic linkages at subsites -1 to +1 on molecule 1 and subsites -2 to -1 on molecule 2 significantly changed compared with that of each molecule of wild-type-acarbose complex to adopt the structural change of subsite +2. These structural and biochemical data suggest that substrate binding in the active site of CGTase is critically affected by the carbohydrate/aromatic stacking interaction with Tyr100 at the catalytic subsite -1 and that this effect is likely a result of cooperation between Tyr100 and Phe259 through stacking interaction with substrate at subsite +2.