A β-mannanase with a lysozyme-like fold and a novel molecular catalytic mechanism

The enzymatic cleavage of β-1,4-mannans is achieved by endo-β-1,4-mannanases, enzymes involved in germination of seeds and microbial hemicellulose degradation, and which have increasing industrial and consumer product applications. β- Mannanases occur in a range of families of the CAZy sequence-based glycoside hydrolase (GH) classification scheme including families 5, 26, and 113. In this work we reveal that β- mannanases of the newly described GH family 134 differ from other mannanase families in both their mechanism and tertiary structure. A representative GH family 134 endo-β-1,4-mannanase from a Streptomyces sp. displays a fold closely related to that of hen egg white lysozyme but acts with inversion of stereochemistry. A Michaelis complex with mannopentaose, and a product complex with mannotriose, reveal ligands with pyranose rings distorted in an unusual inverted chair conformation. Ab initio quantum mechanics/molecular mechanics metadynamics quantified the energetically accessible ring conformations and provided evidence in support of a 1C4 → 3H4 ‡ → 3S1 conformational itinerary along the reaction coordinate. This work, in concert with that on GH family 124 cellulases, reveals how the lysozyme fold can be co-opted to catalyze the hydrolysis of different polysaccharides in a mechanistically distinct manner

Enzymatic conversion of β-mannans: Analysing, evaluating and modifying transglycosylation properties of glycoside hydrolases

Retaining glycoside hydrolases are enzymes that catalyse the breakdown down of glycans through hydrolysis. Due to the double-replacement mechanism of the retaining glycoside hydrolases (GHs), which form an intermediate with part of the glycan covalently attached to the enzyme, some GHs are able to catalyse synthesis reactions called transglycosylation. In transglycosylation reactions a hydroxyl-containing molecule (acceptor), other than water, acts as a nucleophile which releases the glycan moiety from the covalent intermediate while forming a new glycoside (transglycosylation product). The transglycosylation reaction can be used to transform renewable starting materials such as plant hemicellulose to valuable products, which is discussed in the thesis. The work presented in the thesis have explored how GHs interact with glycans and how different aspects of transglycosylation reactions affect the final yield of transglycosylation products. The presented work explores how the open active site structure of two GH26 β-mannanases have made them well adapted to act on heavily galactosylated hemicellulosic β-mannan polysaccharides (Paper I and II). In addition Paper I and II explore how substitutions of amino acids in glycan interacting subsites can lead to changes in catalytic properties and how the two GH26 β-mannanases productively interacts with oligosaccharides. The work also examines how variants of GHs can have improved transglycosylation capacity compared to their wildtype counterparts (Paper III and V). It investigates how the elimination of saccharide interactions in the +2 subsites can lead to improved transglycosylation capacity in a variant of the GH5 β-mannanase TrMan5A (PaperIII). The TrMan5A variant displayed greatly improved transglycosylation capacity at the early timepoints. Observed secondary (product) hydrolysis at later times highlighted the importance of analysing prolonged reaction times to determine suitable reaction termination. Paper III also demonstrated how enzyme synergy can lead to increased transglycosylation yields, when TrMan5A and a guar α-galactosidase was used in co-incubations where a galactomannan was used as the glycosyl donor. α-Galactosidases were further studied in Paper IV, where thetransglycosylation capacity of two different α-galactosidases were explored with different glycosyl donors and acceptor molecules. The study showed that the guar α-galactosidase was able to utilise a wide variety of acceptor molecules and glycosyl donors, further expanding potential transglycosylation products that may be produced with the enzyme. Paper IV further highlights the negative effects secondary hydrolysis may have on transglycosylation yields. The presented work also shows how targeting highly conserved residues within a glycoside hydrolase family can be used to quickly generate GH variants with improved transglycosylation capacity compared to the wild type GH (Paper V). The method relies on protein sequence data and does not require structural knowledge of the target enzyme. Furthermore, the method generates few variants (evolution (100s to 1000s) while it appears to be generally applicable as it was successfully applied to six different GH families covering varying specificities. Improvements was, in part, indicated to be associated with reduced secondary hydrolysis in several of the six GH families in the study. The results presented in the thesis have expanded the knowledge of different factors that affects and can be manipulated in order to improve the transglycosylation capacity in retaining glycoside hydrolases. The work presented in the thesis will help further enzymatic synthesis approaches utilising renewable raw-materials

Mannan-hydrolysis by hemicellulases: enzyme-polysaccharide interaction of a modular beta-mannanase

The enzymatic degradation of plant polysaccharides is a process of fundamental importance in nature which involves a wide range of enzymes. In this work, the structure and function of hemicellulose-degrading enzymes was investigated. The focus was on a beta-mannanase (TrMan5A) produced by the filamentous fungus Trichoderma reesei. This enzyme is composed of a catalytic module and a carbohydrate-binding module (CBM). In this thesis, the enzyme-polysaccharide interaction in both these modules was investigated. The results demonstrate that the CBM of TrMan5A is important for hydrolysis of complex mannan substrates containing cellulose. Furthermore, the increase in activity could be linked to binding of the CBM to the complex substrate. Binding studies revealed that the CBM binds to cellulose, but not to mannan. Studies of the enzyme/polysaccharide interaction in the active site cleft of the catalytic module of TrMan5A showed that a mutant of Arg 171 displayed activity in the same range as the wild-type enzyme toward polymeric substrates. However, the Arg171 mutant was impaired in hydrolysis of small substrates. Interestingly, this mutant also appears to have a more alkaline activity pH-optimum than the wild-type. The low or abolished activity observed with mutants of the predicted catalytic glutamates (Glu169 and Glu276) support their importance in hydrolysis. In addition to TrMan5A, the properties of a beta-mannanase (MeMan5A) from blue mussel and a beta-mannosidase (AnMan2A) from Aspergillus niger, were studied in this work. Investigations on the catalytic properties of the enzymes showed that all three enzymes are capable of degrading polymeric mannan. Furthermore, analysis by transmission electron microscopy revealed that TrMan5A and AnMan2A degrades highly crystalline mannan. Degradation of glucomannan and galactoglucomannan by several polysaccharide-degrading enzymes shows that these substrates can be hydrolysed by both mannoside- and glucoside-hydrolases. Furthermore, the results presented show that cellulases potentially are able to hydrolyse other components in the plant cell wall. Altogether, the results presented demonstrates the need to use complex substrates in order to reveal the mechanisms of plant polysaccharide degradation. In conclusion, this work has shown that the enzyme/polysaccharide interaction in the two modules of TrMan5A is important in determining the overall enzymatic activity and specificity

Mannosidase mechanism : at the intersection of conformation and catalysis

Mannosidases are a diverse group of enzymes that are important in the biological processing of mannose-containing polysaccharides and complex glycoconjugates. They are found in 12 of the >160 sequence-based glycosidase families. We discuss evidence that nature has evolved a small set of common mechanisms that unite almost all of these mannosidase families. Broadly, mannosidases (and the closely related rhamnosidases) perform catalysis through just two conformations of the oxocarbenium ion-like transition state: a B2,5 (or enantiomeric 2,5B) boat and a 3H4 half-chair. This extends to a new family (GT108) of GDPMan-dependent β-1,2-mannosyltransferases/phosphorylases that perform mannosyl transfer through a boat conformation as well as some mannosidases that are metalloenzymes and require divalent cations for catalysis. Yet, among this commonality lies diversity. New evidence shows that one unique family (GH99) of mannosidases use an unusual mechanism involving anchimeric assistance via a 1,2-anhydro sugar (epoxide) intermediate

Discovering novel carbohydrate-active enzymes in the cellulosome of anaerobic bacteria

Tese de Doutoramento em Ciências Veterinárias, especialidade em Ciências Biológicas e BiomédicasCarbohydrate-active enzymes (CAZymes) include a range of enzymes that, in nature, make, break or modify glycosidic bonds. CAZymes act on highly recalcitrant polysaccharides, such as cellulose and hemicellulose, and often exhibit a modular architecture including catalytic domains fused through flexible linker regions to non-catalytic domains such as carbohydrate-binding modules (CBMs). In some anaerobic bacteria these enzymes can associate in high molecular mass multi-enzyme complexes termed cellulosomes. Cellulosomal organisms express a vast repertoire of plant cell wall degrading enzymes and constitute a promising source for the discovery of novel CAZymes. Presently, an exponential accumulation of genomic and metagenomic information is observed while the identification of the biological role of both genes and proteins of unknown function is sorely lacking. In addition, for most of the known CAZymes, structure and/or biochemical characterization is missing. In this study we have developed innovative approaches for the discovery of novel CAZymes in cellulosomal bacteria and provide a detailed biochemical characterization of some of those enzymes. A high-throughput platform was designed for cloning, expression and production of recombinant cellulosomal proteins in Escherichia coli, aiming at looking for novel cellulosomal CAZymes encoded in the genomes of Clostridium thermocellum and Ruminococcus flavefaciens. As a result, a series of novel prokaryotic expression vectors (pHTP) were constructed to allow ligation-independent cloning with high levels of soluble recombinant protein production. In addition, to allow total automation of the procedure, both novel cell culture media and protein purification methods have been established. The platform allowed the production of 184 cellulosomal proteins of unknown function that after the implementation of an enzyme discovery screen lead to the discovery of a novel family of α-Larabinofuranosidases. In order to achieve recombinant soluble expression in E. coli, novel fusion tags were designed and incorporated into pHTP-derivatives. Both Rf1 and Rf47 tags, derived from cellulosomal components, were shown to display a high capacity to enhance protein solubility, as fusion proteins containing both these tags were expressed at high levels and in the soluble form in E. coli. CBMs were confirmed to affect the catalytic activity of appended CAZymes, as it was illustrated by the CBM32 of CtMan5A. This work revealed that members of family 35 CBM have the capacity to bind β-mannose-containing polymers. The biochemical characterization of PL1A, PL1B and PL9 reported here describes the pectinolytic activity expressed by C. thermocellum cellulosome. These enzymes are appended to CBMs that display considerable ligand promiscuity. The application of β- glucanases in animal feed supplementation was tested either in the free state or while associated in mini-cellulosomes. This study revealed that β-1,3-1,4-glucanases and not β-1,4-glucanases are necessary to improve the nutritive value of barley-based diets for broilers. In addition, it was shown that mini-cellulosomes designed to improve the efficacy of exogenous enzymes used for feed supplementation require an effective mechanism to protect linker regions from proteolytic cleavage.RESUMO - Descoberta de novas enzimas celulossomais de bactérias anaeróbias que degradam hidratos de carbono - As enzimas que na natureza degradam os hidratos de carbono (CAZymes) são capazes de construir, quebrar ou modificar ligações glicosídicas. Estas enzimas actuam sobre polissacáridos complexos e recalcitrantes, como a celulose e a hemicelulose, e apresentam geralmente uma estrutura modular, podendo incluir módulos catalíticos fundidos através de sequências de ligação a domínios não catalíticos, sendo os mais comuns os módulos de ligação a hidratos de carbono (CBMs). Em algumas bactérias anaeróbias, estas enzimas podem associar-se em complexos multi-enzimáticos de elevada massa molecular designados de celulossomas. Os organismos que produzem estes complexos apresentam um vasto repertório de enzimas envolvidas na degradação da parede celular vegetal e constituem um bom ponto de partida para a descoberta de novas CAZymes. Actualmente, verifica-se uma crescente acumulação de informação genómica e metagenómica a um ritmo superior à capacidade de identificação da função biológica de uma plêiade de genes e proteínas de funções desconhecidas. Para além disso, para a maioria das CAZymes já conhecidas, não foi ainda efectuada uma caracterização estrutural e/ou bioquímica. Neste estudo foram desenvolvidas metodologias inovadoras para a descoberta de novas CAZymes em bactérias celulossomais, bem como se procedeu a uma caracterização bioquímica detalhada para algumas destas enzimas. Desenvolveu-se uma plataforma de alta capacidade para a clonagem, expressão e produção de proteínas celulossomais recombinantes em Escherichia coli, tendo como objectivo descobrir novas CAZymes codificadas nos genomas de Clostridium thermocellum e Ruminococcus flavefaciens. Como resultado, foi construída uma nova série de vectores de expressão (pHTP) a fim de sustentarem um método de clonagem independente de ligação. Para possibilitar a total automatização do processo foram desenvolvidos novos meios de cultura celulares e métodos de purificação de proteínas adaptados a um esquema de produção de alta capacidade. A pesquisa de novas enzimas nos módulos celulossomais de função desconhecida possibilitou a descoberta de uma nova α-L-arabinofuranosidase em R. flavefaciens, que se constitui como a enzima fundadora de uma nova família de CAZymes. A fim de potenciar a solubilidade de proteínas recombinantes em E. coli, foram desenhadas novas tags de fusão, as quais foram incorporadas em vectores derivados do pHTP. Tanto as tags Rf1 como Rf47, derivadas de componentes celulossomais, mostraram possuir uma capacidade elevada para potenciar a solubilidade de proteínas, uma vez que as proteínas de fusão contendo quer uma quer outra destas tags foram produzidas na forma solúvel em níveis mais elevados do que com parceiros de fusão anteriormente descritos. Confirmou-se que os CBMs afectam a actividade catalítica das CAZymes associadas, tal como ilustrado pelo CBM32 da CtMan5A. Este trabalho forneceu indicações de que os CBMs membros da família 35 têm a capacidade de se ligarem a polímeros de β-manose. A caracterização bioquímica das PL1A, PL1B e PL9 aqui descrita constituiu o primeiro relato de actividade pectinolítica no celulossoma de C. thermocellum. Estas enzimas podem estar associadas a CBMs que revelam pouca especificidade de ligação aos substratos. Testou-se a aplicação de β-glucanases na suplementação alimentar animal, tanto como enzimas isoladas, como associadas em mini-celulossomas. Os dados apresentados aqui revelam que são as β-1,3-1,4-glucanases e não as β-1,4-glucanases as enzimas responsáveis por melhorar o valor nutritivo de dietas à base de cevada para frangos. Por outro lado, os resultados mostram que a eficácia dos mini-celulossomas para melhorar o desempenho das enzimas exógenas usadas na suplementação alimentar requer um mecanismo eficaz para proteger as regiões de ligação entre os componentes celulossomais da degradação por proteases

Glycoside hydrolase from the GH76 family indicates that marine Salegentibacter sp. Hel_I_6 consumes alpha-mannan from fungi

Microbial glycan degradation is essential to global carbon cycling. The marine bacterium Salegentibacter sp. Hel_I_6 (Bacteroidota) isolated from seawater off Helgoland island (North Sea) contains an α-mannan inducible gene cluster with a GH76 family endo-α-1,6-mannanase (ShGH76). This cluster is related to genetic loci employed by human gut bacteria to digest fungal α-mannan. Metagenomes from the Hel_I_6 isolation site revealed increasing GH76 gene frequencies in free-living bacteria during microalgae blooms, suggesting degradation of α-1,6-mannans from fungi. Recombinant ShGH76 protein activity assays with yeast α-mannan and synthetic oligomannans showed endo-α-1,6-mannanase activity. Resolved structures of apo-ShGH76 (2.0 Å) and of mutants co-crystalized with fungal mannan-mimicking α-1,6-mannotetrose (1.90 Å) and α-1,6-mannotriose (1.47 Å) retained the canonical (α/α)6 fold, despite low identities with sequences of known GH76 structures (GH76s from gut bacteria: <27%). The apo-form active site differed from those known from gut bacteria, and co-crystallizations revealed a kinked oligomannan conformation. Co-crystallizations also revealed precise molecular-scale interactions of ShGH76 with fungal mannan-mimicking oligomannans, indicating adaptation to this particular type of substrate. Our data hence suggest presence of yet unknown fungal α-1,6-mannans in marine ecosystems, in particular during microalgal blooms

β-Mannan degradation by gut bacteria - Characterization of β-mannanases from families GH5 and GH26

The human gut flora is important for our well-being. The gut bacteria are able to degrade and metabolize complex carbohydrates. Examples of such carbohydrates are β-mannans. β-Mannans consist of a backbone of β-1,4-linked mannose units and are present in e.g. the endosperm of legumes such as guar or carob. The composition of the β-mannan varies with the plant source. Carob and guar β-mannans are substituted with α-1,6-linked galactose units and are soluble in water. They make a viscous solution that is used e.g. as food thickener. It is known that guar gum galactomannan can be fermented by the human gut flora. The main enzymes that hydrolyze β-mannan backbones are called β-mannanases. β-Mannanases have been studied and characterized from a range of environments. However, β-mannanases have yet not been studied from the gut flora to any significant extent. This thesis focuses on β-mannanases from families GH5 and GH26 from the gut bacteria Bacteroides and Bifidobacterium. In Paper I, we have studied the effect of galactomannan on metabolic markers and four bacterial genera in rats fed with guar gum. Guar gum of different viscosities was tested. We found that Bifidobacterium counts were increased when the rats were fed guar gum, regardless of viscosity, while the number of Bacteroides was not different from the control. In Papers II-IV we characterized four enzymes from these genera possibly involved in the degradation of guar gum and other mannans. We characterized BaMan26A, a GH26 β-mannanase from Bifidobacterium adolescentis in Paper II and a GH5 β-mannanase, BlMan5_8, from Bifidobacterium animalis subsp. lactis in Paper IV. In Paper III we characterized two GH26 β-mannanases, BoMan26A and BoMan26B, from Bacteroides ovatus that are encoded by a polysaccharide utilization locus. A crystal structure of BoMan26A displayed the (α/β)8 fold of clan GH-A enzymes and a substrate binding cleft. The four β-mannanases were found to vary in product profile and fine-tuned substrate specificity within the group of β-mannans. While BlMan5_8 produced oligosaccharides of varying length from β-mannan, BoMan26A produced almost exclusively mannobiose from β-mannan. BoMan26B produced mainly mannobiose, while BaMan26A produced mannotriose as a major product. BlMan5_8 is predicted to be secreted, while BoMan26B and BaMan26A appear to be anchored to the cell. BaMan26A is predicted to be located in the periplasmic space. This work gives further insight in the molecular details of β-mannan catabolism in the gut

Crystal structure and substrate interactions of an unusual fungal non-CBM carrying GH26 endo-β-mannanase from Yunnania penicillata

Endo-β(1 → 4)-mannanases (endomannanases) catalyse degradation of β-mannans, an abundant class of plant polysaccharides. This study investigates structural features and substrate binding of YpenMan26A, a non-CBM carrying endomannanase from Yunnania penicillata. Structural and sequence comparisons to other fungal family GH26 endomannanases showed high sequence similarities and conserved binding residues, indicating that fungal GH26 endomannanases accommodate galactopyranosyl units in the −3 and −2 subsites. Two striking amino acid differences in the active site were found when the YpenMan26A structure was compared to a homology model of Wsp.Man26A from Westerdykella sp. and the sequences of nine other fungal GH26 endomannanases. Two YpenMan26A mutants, W110H and D37T, inspired by differences observed in Wsp.Man26A, produced a shift in how mannopentaose bound across the active site cleft and a decreased affinity for galactose in the −2 subsite, respectively, compared to YpenMan26A. YpenMan26A was moreover found to have a flexible surface loop in the position where PansMan26A from Podospora anserina has an α-helix (α9) which interacts with its family 35 CBM. Sequence alignment inferred that the core structure of fungal GH26 endomannanases differ depending on the natural presence of this type of CBM. These new findings have implications for selecting and optimising these enzymes for galactomannandegradation