Elucidating Sequence and Structural Determinants of Carbohydrate Esterases for Complete Deacetylation of Substituted Xylans

| openaire: EC/H2020/648925/EU//BHIVEAcetylated glucuronoxylan is one of the most common types of hemicellulose in nature. The structure is formed by a β-(1→4)-linked D-xylopyranosyl (Xylp) backbone that can be substituted with an acetyl group at O-2 and O-3 positions, and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Acetyl xylan esterases (AcXE) that target mono-or doubly acetylated Xylp are well characterized; however, the previously studied AcXE from Flavobacterium johnsoniae (FjoAcXE) was the first to remove the acetyl group from 2-O-MeGlcpA-3-O-acetyl-substituted Xylp units, yet structural characteristics of these enzymes remain unspecified. Here, six homologs of FjoAcXE were produced and three crystal structures of the enzymes were solved. Two of them are complex structures, one with bound MeGlcpA and another with acetate. All homologs were confirmed to release acetate from 2-O-MeGlcpA-3-O-acetyl-substituted xylan, and the crystal structures point to key structural elements that might serve as defining features of this unclassified carbohydrate esterase family. Enzymes comprised two domains: N-terminal CBM domain and a C-terminal SGNH domain. In FjoAcXE and all studied homologs, the sequence motif around the catalytic serine is Gly-Asn-Ser-Ile (GNSI), which differs from other SGNH hydrolases. Binding by the MeGlcpA-Xylp ligand is directed by positively charged and highly conserved residues at the interface of the CBM and SGNH domains of the enzyme.Peer reviewe

In vivo and in vitro FMN prenylation and (de)carboxylase activation under aerobic conditions

Prenylated FMN (prFMN) is a newly discovered redox cofactor required for activity of the large family of reversible UbiD (de)carboxylases involved in biotransformation of aromatic, heteroaromatic, and unsaturated aliphatic acids (White et al., 2015). Despite the growing demand for decarboxylases in the pulp/paper industry and in forest biorefineries, the vast majority of UbiD-like decarboxylases remain uncharacterized. Functional characterization of the novel UbiD decarboxylases is hindered by the lack of prFMN generating system. prFMN cofactor is synthesized by the UbiX family of FMN prenyltransferases, which use reduced FMN as substrate under anaerobic conditions and dimethylallyl-monophosphate (DMAP) as the prenyl group donor. Here, we report the in vivo and in vitro biosynthesis of prFMN and UbiD activation under aerobic conditions. For in vivo biosynthesis, we used newly discovered UbiX proteins from Salmonella typhimurium and Klebsiella pneumonia, which activated ferulic acid UbiD decarboxylase Fdc1 from Aspergillus niger under aerobic conditions (0.5-1.5 U/mg). For in vitro biosynthesis of prFMN and UbiD activation, we established a one-pot enzyme cascade system that uses prenol, polyphosphate, formate, and riboflavin as starting substrates and (re)generates DMAP, ATP, FMN and NADH. The system contains 6 different enzymes: prenol kinase, polyphosphate kinase, formate dehydrogenase, FMN reductase, riboflavin kinase and FMN prenyltransferase. Under aerobic conditions, this system showed up to 80% conversion of FMN to prFMN and generated active Fdc1 decarboxylase (0.2-1 U/mg). Thus, both systems represent robust approaches for in vivo and in vitro prFMN biosynthesis and UbiD activation under aerobic conditions. The developed FMN prenylation systems will facilitate the exploration and biochemical characterization of UbiD-like decarboxylases and their applications in biocatalysis. Please click Additional Files below to see the full abstract

Refining and mining the phylogeny of Glycoside Hydrolase Family 74 via structure-function analysis

Sustained interest in the use of carbohydrates from plant cell walls, coupled with the advancement of high-throughput (meta)genomic sequencing, has led to the discovery of an overwhelming number of predicted carbohydrate-active enzymes (CAZymes) in the last decade. The CAZy database provides a powerful framework for the study of CAZymes, including Glycoside Hydrolases (GHs), by enabling the prediction of key enzyme features such as 3-D fold, catalytic residues, catalytic mechanism, and – with certain limitations – substrate specificity. Refined phylogenetic analyses contribute to increasing the accuracy of predictions by further clustering proteins into sub-families (1, 2). However, reliable prediction of substrate specificity for newly discovered GHs remains a challenge due to a general lack of in-depth biochemical and structural characterization across the existing phylogenetic diversity. Glycoside Hydrolase family 74 (GH74) comprises endo-glucanases, many of which have predominant activity toward xyloglucan, a highly branched plant cell wall matrix glycan. To better delineate overall substrate specificity, backbone cleavage position, and endo-dissociative vs. endo-processive hydrolytic modes, a broad-based structure-function analysis of GH74 guided by molecular phylogeny was performed. Seven sub-families were discerned, which grouped nearly 40% of the current \u3e300 GH74 sequences in the public CAZy database. Thirty one GH74 members were targeted for further investigation based on their phylogenetic position and unique primary structural features identified during manual curation. The biochemical characterization of 18 recombinant GH74s revealed key sequence features governing xyloglucan backbone cleavage sites and highlighted clear phylogenetic differences between endo-dissociative and endo-processive enzymes. Commensurate with previous studies (3), site-directed mutagenesis of key active-site tryptophan residues defined their essential contributions to processivity on the soluble polysaccharide substrate. Six new GH74 tertiary structures (apo and/or in complex with xylogluco-oligosaccharides) were determined that further resolved the contribution of active-site loops in modulating the size of oligosaccharide products released by individual subfamily members. Refining the correlation between phylogeny and enzyme structure-function properties in GH74 significantly enhances the prediction of catalytic ability, highlights key steps in the evolution of function in the family, and ultimately informs applications in biomass conversion. 1. Stam MR, Danchin EGJ, Rancurel C, Coutinho PM, Henrissat B. 2006. Dividing the large glycoside hydrolase family 13 into subfamilies: towards improved functional annotations of alpha-amylase-related proteins. Protein Engineering Design & Selection 19:555-562. 2. Aspeborg H, Coutinho PM, Wang Y, Brumer H, Henrissat B. 2012. Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5). Bmc Evolutionary Biology 12. 3. Matsuzawa T, Saito Y, Yaoi K. 2014. Key amino acid residues for the endo-processive activity of GH74 xyloglucanase. FEBS Lett 588:1731-8

Elucidating Sequence and Structural Determinants of Carbohydrate Esterases for Complete Deacetylation of Substituted Xylans

Acetylated glucuronoxylan is one of the most common types of hemicellulose in nature. The structure is formed by a β-(1→4)-linked D-xylopyranosyl (Xylp) backbone that can be substituted with an acetyl group at O-2 and O-3 positions, and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Acetyl xylan esterases (AcXE) that target mono- or doubly acetylated Xylp are well characterized; however, the previously studied AcXE from Flavobacterium johnsoniae (FjoAcXE) was the first to remove the acetyl group from 2-O-MeGlcpA-3-O-acetyl-substituted Xylp units, yet structural characteristics of these enzymes remain unspecified. Here, six homologs of FjoAcXE were produced and three crystal structures of the enzymes were solved. Two of them are complex structures, one with bound MeGlcpA and another with acetate. All homologs were confirmed to release acetate from 2-O-MeGlcpA-3-O-acetyl-substituted xylan, and the crystal structures point to key structural elements that might serve as defining features of this unclassified carbohydrate esterase family. Enzymes comprised two domains: N-terminal CBM domain and a C-terminal SGNH domain. In FjoAcXE and all studied homologs, the sequence motif around the catalytic serine is Gly-Asn-Ser-Ile (GNSI), which differs from other SGNH hydrolases. Binding by the MeGlcpA-Xylp ligand is directed by positively charged and highly conserved residues at the interface of the CBM and SGNH domains of the enzyme

Elucidating Sequence and Structural Determinants of Carbohydrate Esterases for Complete Deacetylation of Substituted Xylans

Acetylated glucuronoxylan is one of the most common types of hemicellulose in nature. The structure is formed by a β-(1→4)-linked D-xylopyranosyl (Xylp) backbone that can be substituted with an acetyl group at O-2 and O-3 positions, and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Acetyl xylan esterases (AcXE) that target mono- or doubly acetylated Xylp are well characterized; however, the previously studied AcXE from Flavobacterium johnsoniae (FjoAcXE) was the first to remove the acetyl group from 2-O-MeGlcpA-3-O-acetyl-substituted Xylp units, yet structural characteristics of these enzymes remain unspecified. Here, six homologs of FjoAcXE were produced and three crystal structures of the enzymes were solved. Two of them are complex structures, one with bound MeGlcpA and another with acetate. All homologs were confirmed to release acetate from 2-O-MeGlcpA-3-O-acetyl-substituted xylan, and the crystal structures point to key structural elements that might serve as defining features of this unclassified carbohydrate esterase family. Enzymes comprised two domains: N-terminal CBM domain and a C-terminal SGNH domain. In FjoAcXE and all studied homologs, the sequence motif around the catalytic serine is Gly-Asn-Ser-Ile (GNSI), which differs from other SGNH hydrolases. Binding by the MeGlcpA-Xylp ligand is directed by positively charged and highly conserved residues at the interface of the CBM and SGNH domains of the enzyme

Identification and characterization of a large family of superbinding bacterial SH2 domains

Src homology 2 (SH2) domains play a critical role in signal transduction in mammalian cells by binding to phosphorylated Tyr (pTyr). Apart from a few isolated cases in viruses, no functional SH2 domain has been identified to date in prokaryotes. Here we identify 93 SH2 domains from Legionella that are distinct in sequence and specificity from mammalian SH2 domains. The bacterial SH2 domains are not only capable of binding proteins or peptides in a Tyr phosphorylation-dependent manner, some bind pTyr itself with micromolar affinities, a property not observed for mammalian SH2 domains. The Legionella SH2 domains feature the SH2 fold and a pTyr-binding pocket, but lack a specificity pocket found in a typical mammalian SH2 domain for recognition of sequences flanking the pTyr residue. Our work expands the boundary of phosphotyrosine signalling to prokaryotes, suggesting that some bacterial effector proteins have acquired pTyr-superbinding characteristics to facilitate bacterium-host interactions

Structure of SAICAR synthase from Thermotoga maritima at 2.2 Å reveals an unusual covalent dimer

The crystal structure of phophoribosylaminoimidazole-succinocarboxamide or SAICAR synthase from T. maritima at 2.2 Å revealed an unusual covalent dimer

Structural characterization of the family GH115 alpha-glucuronidase from Amphibacillus xylanus yields insight into its coordinated action with alpha-arabinofuranosidases

The coordinated action of carbohydrate-active enzymes has mainly been evaluated for the purpose of complete saccharification of plant biomass (lignocellulose) to sugars. By contrast, the coordinated action of accessory hemicellulases on xylan debranching and recovery is less well characterized. Here, the activity of two family GH115 alpha-glucuronidases (SdeAgu115A from Saccharophagus degradans, and AxyAgu115A from Amphibacillus xylanus) on spruce arabinoglucuronoxylan (AGX) was evaluated in combination with an alpha-arabinofuranosidase from families GH51 (AniAbf51A, aka E-AFASE from Aspergillus niger) and GH62 (SthAbf62A from Streptomyces thermoviolaceus). The alpha-arabinofuranosidases boosted (methyl)-glucuronic acid release by SdeAgu115A by approximately 50 % and 30 %, respectively. The impact of the alpha-arabinofuranosidases on AxyAgu115A activity was comparatively low, motivating its structural characterization. The crystal structure of AxyAgu115A revealed increased length and flexibility of the active site loop compared to SdeAgu115A. This structural difference could explain the ability of AxyAgu115A to accommodate more highly substituted arabinoglucuronoxylan, and inform enzyme selections for improved AGX recovery and use.Peer reviewe

Structural and molecular rationale for the diversification of resistance mediated by the Antibiotic_NAT family

The environmental microbiome harbors a vast repertoire of antibiotic resistance genes (ARGs) which can serve as evolutionary predecessors for ARGs found in pathogenic bacteria, or can be directly mobilized to pathogens in the presence of selection pressures. Thus, ARGs from benign environmental bacteria are an important resource for understanding clinically relevant resistance. Here, we conduct a comprehensive functional analysis of the Antibiotic_NAT family of aminoglycoside acetyltransferases. We determined a pan-family antibiogram of 21 Antibiotic_NAT enzymes, including 8 derived from clinical isolates and 13 from environmental metagenomic samples. We find that environment-derived representatives confer high-level, broad-spectrum resistance, including against the atypical aminoglycoside apramycin, and that a metagenome-derived gene likely is ancestral to an aac(3) gene found in clinical isolates. Through crystallographic analysis, we rationalize the molecular basis for diversification of substrate specificity across the family. This work provides critical data on the molecular mechanism underpinning resistance to established and emergent aminoglycoside antibiotics and broadens our understanding of ARGs in the environment

Large-scale screening of preferred interactions of human src homology-3 (SH3) domains using native target proteins as affinity ligands

The Src Homology-3 (SH3) domains are ubiquitous protein modules that mediate important intracellular protein interactions via binding to short proline-rich consensus motifs in their target proteins. The affinity and specificity of such core SH3-ligand contacts are typically modest, but additional binding interfaces can give rise to stronger and more specific SH3-mediated interactions. To understand how commonly such robust SH3 interactions occur in the human protein interactome, and to identify these in an unbiased manner we have expressed 324 predicted human SH3 ligands as full-length proteins in mammalian cells, and screened for their preferred SH3 partners using a phage display-based approach. This discovery platform contains an essentially complete repertoire of the ∼300 human SH3 domains, and involves an inherent binding threshold that ensures selective identification of only SH3 interactions with relatively high affinity. Such strong and selective SH3 partners could be identified for only 19 of these 324 predicted ligand proteins, suggesting that the majority of human SH3 interactions are relatively weak, and thereby have capacity for only modest inherent selectivity. The panel of exceptionally robust SH3 interactions identified here provides a rich source of leads and hypotheses for further studies. However, a truly comprehensive characterization of the human SH3 interactome will require novel high-throughput methods based on function instead of absolute binding affinity