RESUMO
Gum arabic (GA) is widely used as an emulsion stabilizer and edible coating and consists of a complex carbohydrate moiety with a rhamnosyl-glucuronate group capping the non-reducing ends. Enzymes that can specifically cleave the glycosidic chains of GA and modify their properties are valuable for structural analysis and industrial application. Cryogenic X-ray crystal structure of GA-specific L-rhamnose-α-1,4-D-glucuronate lyase from Fusarium oxysporum (FoRham1), belonging to the polysaccharide lyase (PL) family 42, has been previously reported. To determine the specific reaction mechanism based on its hydrogen-containing enzyme structure, we performed joint X-ray/neutron crystallography of FoRham1. Large crystals were grown in the presence of L-rhamnose (a reaction product), and neutron and X-ray diffraction datasets were collected at room temperature at 1.80 and 1.25 Å resolutions, respectively. The active site contained L-rhamnose and acetate, the latter being a partial analog of glucuronate. Incomplete H/D exchange between Arg166 and acetate suggested that a strong salt-bridge interaction was maintained. Doubly deuterated His105 and deuterated Tyr150 supported the interaction between Arg166 and the acetate. The unique hydrogen-rich environment functions as a charge neutralizer for glucuronate and stabilizes the oxyanion intermediate. The NE2 atom of His85 was deprotonated and formed a hydrogen bond with the deuterated O1 hydroxy of L-rhamnose, indicating the function of His85 as the base/acid catalyst for bond cleavage via ß-elimination. Asp83 functions as a pivot between the two catalytic histidine residues by bridging them. This His-His-Asp structural motif is conserved in the PL 24, 25, and 42 families.
Assuntos
Fusarium , Polissacarídeo-Liases , Humanos , Acetatos , Cristalografia por Raios X , Ácido Glucurônico/química , Hidrogênio , Liases , Polissacarídeo-Liases/química , Ramnose/química , Fusarium/enzimologiaRESUMO
Para-hydroxybenzoate hydroxylase (PHBH) is a group A flavoprotein monooxygenase that hydroxylates p-hydroxybenzoate to protocatechuate (PCA). Despite intensive studies of Pseudomonas aeruginosa p-hydroxybenzoate hydroxylase (PaPobA), the catalytic reactions of extremely diverse putative PHBH isozymes remain unresolved. We analyzed the phylogenetic relationships of known and predicted PHBHs and identified eight divergent clades. Clade F contains a protein that lacks the critical amino acid residues required for PaPobA to generate PHBH activity. Among proteins in this clade, Xylophilus ampelinus PobA (XaPobA) preferred PCA as a substrate and is the first known natural PCA 5-hydroxylase (PCAH). Crystal structures and kinetic properties revealed similar mechanisms of substrate carboxy group recognition between XaPobA and PaPobA. The unique Ile75, Met72, Val199, Trp201, and Phe385 residues of XaPobA form the bottom of a hydrophobic cavity with a shape that complements the 3-and 4-hydroxy groups of PCA and its binding site configuration. An interaction between the δ-sulfur atom of Met210 and the aromatic ring of PCA is likely to stabilize XaPobA-PCA complexes. The 4-hydroxy group of PCA forms a hydrogen bond with the main chain carbonyl of Thr294. These modes of binding constitute a novel substrate recognition mechanism that PaPobA lacks. This mechanism characterizes XaPobA and sheds light on the diversity of catalytic mechanisms of PobA-type PHBHs and group A flavoprotein monooxygenases.
Assuntos
4-Hidroxibenzoato-3-Mono-Oxigenase , Pseudomonas , 4-Hidroxibenzoato-3-Mono-Oxigenase/metabolismo , Sítios de Ligação , Flavoproteínas/genética , Flavoproteínas/metabolismo , Cinética , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Filogenia , Pseudomonas/enzimologia , Pseudomonas/metabolismo , Xylophilus/enzimologiaRESUMO
L-Arabinose (L-Ara) is a plant-specific sugar found in cell wall polysaccharides, proteoglycans, glycoproteins, and small glycoconjugates, which play physiologically important roles in cell proliferation and other essential cellular processes. L-Ara is synthesized as UDP-L-arabinose (UDP-L-Ara) from UDP-xylose (UDP-Xyl) by UDP-Xyl 4-epimerases (UXEs), a type of de novo synthesis of L-Ara unique to plants. In Arabidopsis, the Golgi-localized UXE AtMUR4 is the main contributor to UDP-L-Ara synthesis. However, cytosolic bifunctional UDP-glucose 4-epimerases (UGEs) with UXE activity, AtUGE1, and AtUGE3 also catalyze this reaction. For the present study, we first examined the physiological importance of bifunctional UGEs in Arabidopsis. The uge1 and uge3 mutants enhanced the dwarf phenotype of mur4 and further reduced the L-Ara content in cell walls, suggesting that bifunctional UGEs contribute to UDP-L-Ara synthesis. Through the introduction of point mutations exchanging corresponding amino acid residues between AtUGE1 with high UXE activity and AtUGE2 with low UXE activity, two mutations that increase relative UXE activity of AtUGE2 were identified. The crystal structures of AtUGE2 in complex forms with NAD+ and NAD+/UDP revealed that the UDP-binding domain of AtUGE2 has a more closed conformation and smaller sugar-binding site than bacterial and mammalian UGEs, suggesting that plant UGEs have the appropriate size and shape for binding UDP-Xyl and UDP-L-Ara to exhibit UXE activity. The presented results suggest that the capacity for cytosolic synthesis of UDP-L-Ara was acquired by the small sugar-binding site and several mutations of UGEs, enabling diversified utilization of L-Ara in seed plants.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Parede Celular , Citosol , UDPglucose 4-Epimerase , Açúcares de Uridina Difosfato , Arabidopsis/genética , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Citosol/metabolismo , Citosol/enzimologia , Açúcares de Uridina Difosfato/metabolismo , Parede Celular/metabolismo , UDPglucose 4-Epimerase/genética , UDPglucose 4-Epimerase/metabolismo , Mutação , Uridina Difosfato Xilose/metabolismo , Uridina Difosfato Xilose/genéticaRESUMO
Glutathione (GSH) is required for various physiological processes in plants, including redox regulation and detoxification of harmful compounds. GSH also functions as a repository for assimilated sulfur and is actively catabolized in plants. In Arabidopsis, GSH is mainly degraded initially by cytosolic enzymes, γ-glutamyl cyclotransferase, and γ-glutamyl peptidase, which release cysteinylglycine (Cys-Gly). However, the subsequent enzyme responsible for catabolizing this dipeptide has not been identified to date. In the present study, we identified At4g17830 as a Cys-Gly dipeptidase, namely cysteinylglycine peptidase 1 (CGP1). CGP1 complemented the phenotype of the yeast mutant that cannot degrade Cys-Gly. The Arabidopsis cgp1 mutant had lower Cys-Gly degradation activity than the wild type and showed perturbed concentrations of thiol compounds. Recombinant CGP1 showed reasonable Cys-Gly degradation activity in vitro. Metabolomic analysis revealed that cgp1 exhibited signs of severe sulfur deficiency, such as elevated accumulation of O-acetylserine (OAS) and the decrease in sulfur-containing metabolites. Morphological changes observed in cgp1, including longer primary roots of germinating seeds, were also likely associated with sulfur starvation. Notably, At4g17830 has previously been reported to encode an N2-acetylornithine deacetylase (NAOD) that functions in the ornithine biosynthesis. The cgp1 mutant did not show a decrease in ornithine content, whereas the analysis of CGP1 structure did not rule out the possibility that CGP1 has Cys-Gly dipeptidase and NAOD activities. Therefore, we propose that CGP1 is a Cys-Gly dipeptidase that functions in the cytosolic GSH degradation pathway and may play dual roles in GSH and ornithine metabolism.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Citosol , Dipeptidases , Glutationa , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/enzimologia , Glutationa/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Dipeptidases/metabolismo , Dipeptidases/genética , Citosol/metabolismo , Dipeptídeos/metabolismo , Enxofre/metabolismoRESUMO
Mucinolytic bacteria modulate host-microbiota symbiosis and dysbiosis through their ability to degrade mucin O-glycans. However, how and to what extent bacterial enzymes are involved in the breakdown process remains poorly understood. Here we focus on a glycoside hydrolase family 20 sulfoglycosidase (BbhII) from Bifidobacterium bifidum, which releases N-acetylglucosamine-6-sulfate from sulfated mucins. Glycomic analysis showed that, in addition to sulfatases, sulfoglycosidases are involved in mucin O-glycan breakdown in vivo and that the released N-acetylglucosamine-6-sulfate potentially affects gut microbial metabolism, both of which were also supported by a metagenomic data mining analysis. Enzymatic and structural analysis of BbhII reveals the architecture underlying its specificity and the presence of a GlcNAc-6S-specific carbohydrate-binding module (CBM) 32 with a distinct sugar recognition mode that B. bifidum takes advantage of to degrade mucin O-glycans. Comparative analysis of the genomes of prominent mucinolytic bacteria also highlights a CBM-dependent O-glycan breakdown strategy used by B. bifidum.
Assuntos
Ecossistema , Mucinas , Mucinas/metabolismo , Polissacarídeos/metabolismo , Bactérias/metabolismoRESUMO
L-Arabinofuranosides with ß-linkages are present in several plant molecules, such as arabinogalactan proteins (AGPs), extensin, arabinan, and rhamnogalacturonan-II. We previously characterized a ß-L-arabinofuranosidase from Bifidobacterium longum subsp. longum JCM 1217, Bll1HypBA1, which was found to belong to the glycoside hydrolase (GH) family 127. This strain encodes two GH127 genes and two GH146 genes. In the present study, we characterized a GH146 ß-L-arabinofuranosidase, Bll3HypBA1 (BLLJ_1848), which was found to constitute a gene cluster with AGP-degrading enzymes. This recombinant enzyme degraded AGPs and arabinan, which contain Araf-ß1,3-Araf structures. In addition, the recombinant enzyme hydrolyzed oligosaccharides containing Araf-ß1,3-Araf structures but not those containing Araf-ß1,2-Araf and Araf-ß1,5-Araf structures. The crystal structures of Bll3HypBA1 were determined at resolutions up to 1.7 Å. The monomeric structure of Bll3HypBA1 comprised a catalytic (α/α)6 barrel and two ß-sandwich domains. A hairpin structure with two ß-strands was observed in Bll3HypBA1, to extend from a ß-sandwich domain and partially cover the active site. The active site contains a Zn2+ ion coordinated by Cys3-Glu and exhibits structural conservation of the GH127 cysteine glycosidase Bll1HypBA1. This is the first study to report on a ß1,3-specific ß-L-arabinofuranosidase. KEY POINTS: ⢠ß1,3-l-Arabinofuranose residues are present in arabinogalactan proteins and arabinans as a terminal sugar. ⢠ß-l-Arabinofuranosidases are widely present in intestinal bacteria. ⢠Bll3HypBA1 is the first enzyme characterized as a ß1,3-linkage-specific ß-l-arabinofuranosidase.
Assuntos
Bifidobacterium , Glicosídeo Hidrolases , Catálise , CisteínaRESUMO
Glutathione (GSH) functions as a major sulfur repository and hence occupies an important position in primary sulfur metabolism. GSH degradation results in sulfur reallocation and is believed to be carried out mainly by γ-glutamyl cyclotransferases (GGCT2;1, GGCT2;2, and GGCT2;3), which, however, do not fully explain the rapid GSH turnover. Here, we discovered that γ-glutamyl peptidase 1 (GGP1) contributes to GSH degradation through a yeast complementation assay. Recombinant proteins of GGP1, as well as GGP3, showed high degradation activity of GSH, but not of oxidized glutathione (GSSG), in vitro. Notably, the GGP1 transcripts were highly abundant in rosette leaves, in agreement with the ggp1 mutants constantly accumulating more GSH regardless of nutritional conditions. Given the lower energy requirements of the GGP- than the GGCT-mediated pathway, the GGP-mediated pathway could be a more efficient route for GSH degradation than the GGCT-mediated pathway. Therefore, we propose a model wherein cytosolic GSH is degraded chiefly by GGP1 and likely also by GGP3. Another noteworthy fact is that GGPs are known to process GSH conjugates in glucosinolate and camalexin synthesis; indeed, we confirmed that the ggp1 mutant contained higher levels of O-acetyl-l-Ser, a signaling molecule for sulfur starvation, and lower levels of glucosinolates and their degradation products. The predicted structure of GGP1 further provided a rationale for this hypothesis. In conclusion, we suggest that GGP1 and possibly GGP3 play vital roles in both primary and secondary sulfur metabolism.
Assuntos
Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Glucosinolatos/metabolismo , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Enxofre/metabolismoRESUMO
Cytochrome P450 enzymes are promising biocatalysts for industrial use because they catalyze site-selective C-H oxidation and have diverse catalytic reactions and a broad substrate range. In this study, the 2α-hydroxylation activity of CYP154C2 from Streptomyces avermitilis MA-4680T toward androstenedione (ASD) was identified by an in vitro conversion assay. The testosterone (TES)-bound structure of CYP154C2 was solved at 1.42 Å, and this structure was used to design eight mutants, including single, double, and triple mutants, to improve the conversion efficiency. Mutants L88F/M191F and M191F/V285L were found to enhance the conversion rates significantly (i.e., 8.9-fold and 7.4-fold for TES, 46.5-fold and 19.5-fold for ASD, respectively) compared with the wild-type (WT) enzyme while retaining high 2α-position selectivity. The substrate binding affinity of the L88F/M191F mutant toward TES and ASD was enhanced compared with that of WT CYP154C2, supporting the measured increase in the conversion efficiencies. Moreover, the total turnover number and kcat/Km of the L88F/M191F and M191F/V285L mutants increased significantly. Interestingly, all mutants containing L88F generated 16α-hydroxylation products, suggesting that L88 in CYP154C2 plays a vital role in substrate selectivity and that the amino acid corresponding to L88 in the 154C subfamily affects the orientation of steroid binding and substrate selectivity. IMPORTANCE Hydroxylated derivatives of steroids play essential roles in medicine. Cytochrome P450 enzymes selectively hydroxylate methyne groups on steroids, which can dramatically change their polarity, biological activity and toxicity. There is a paucity of reports on the 2α-hydroxylation of steroids, and documented 2α-hydroxylate P450s show extremely low conversion efficiency and/or low regio- and stereoselectivity. This study conducted crystal structure analysis and structure-guided rational engineering of CYP154C2 and efficiently enhanced the conversion efficiency of TES and ASD with high regio- and stereoselectivity. Our results provide an effective strategy and theoretical basis for the 2α-hydroxylation of steroids, and the structure-guided rational design of P450s should facilitate P450 applications in the biosynthesis of steroid drugs.
Assuntos
Sistema Enzimático do Citocromo P-450 , Esteroides , Hidroxilação , Esteroides/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Oxirredução , Testosterona/metabolismo , Especificidade por SubstratoRESUMO
Gum arabic (GA) is widely used as an emulsion stabilizer and coating in several industrial applications, such as foods and pharmaceuticals. GA contains a complex carbohydrate moiety, and the nonreducing ends of the side chains are often capped with l-rhamnose; thus, enzymes that can remove these caps are promising tools for the structural analysis of the carbohydrates comprising GA. In this study, GA-specific l-rhamnose-α-1,4-d-glucuronate lyase from the fungus Fusarium oxysporum 12S (FoRham1) was cloned and characterized. FoRham1 showed the highest amino acid sequence similarity with enzymes belonging to the glycoside hydrolase family 145; however, the catalytic residue on the posterior pocket of the ß-propeller fold protein was not conserved. The catalytic residues of FoRham1 were instead conserved with ulvan lyases belonging to polysaccharide lyase family 24. Kinetic analysis showed that FoRham1 has the highest catalytic efficiency for the substrate α-l-rhamnose-(1â4)-d-glucuronic acid. The crystal structures of ligand-free and α-l-rhamnose-(1â4)-d-glucuronic acid -bound FoRham1 were determined, and the active site was identified on the anterior side of the ß-propeller. The three-dimensional structure of the active site and mutagenesis analysis revealed the detailed catalytic mechanism of FoRham1. Our findings offer a new enzymatic tool for the further analysis of the GA carbohydrate structure and for elucidating its physiological functions in plants. Based on these results, we renamed glycoside hydrolase family 145 as a new polysaccharide lyase family 42, in which FoRham1 is included.
Assuntos
Ácido Glucurônico/metabolismo , Goma Arábica/metabolismo , Polissacarídeo-Liases/metabolismo , Ramnose/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cristalografia por Raios X , Fusarium/enzimologia , Filogenia , Polissacarídeo-Liases/química , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Fructooligosaccharides and their anhydrides are widely used as health-promoting foods and prebiotics. Various enzymes acting on ß-D-fructofuranosyl linkages of natural fructan polymers have been used to produce functional compounds. However, enzymes that hydrolyze and form α-D-fructofuranosyl linkages have been less studied. Here, we identified the BBDE_2040 gene product from Bifidobacterium dentium (α-D-fructofuranosidase and difructose dianhydride I synthase/hydrolase from Bifidobacterium dentium [αFFase1]) as an enzyme with α-D-fructofuranosidase and α-D-arabinofuranosidase activities and an anomer-retaining manner. αFFase1 is not homologous with any known enzymes, suggesting that it is a member of a novel glycoside hydrolase family. When caramelized fructose sugar was incubated with αFFase1, conversions of ß-D-Frup-(2â1)-α-D-Fruf to α-D-Fruf-1,2':2,1'-ß-D-Frup (diheterolevulosan II) and ß-D-Fruf-(2â1)-α-D-Fruf (inulobiose) to α-D-Fruf-1,2':2,1'-ß-D-Fruf (difructose dianhydride I [DFA I]) were observed. The reaction equilibrium between inulobiose and DFA I was biased toward the latter (1:9) to promote the intramolecular dehydrating condensation reaction. Thus, we named this enzyme DFA I synthase/hydrolase. The crystal structures of αFFase1 in complex with ß-D-Fruf and ß-D-Araf were determined at the resolutions of up to 1.76 Å. Modeling of a DFA I molecule in the active site and mutational analysis also identified critical residues for catalysis and substrate binding. The hexameric structure of αFFase1 revealed the connection of the catalytic pocket to a large internal cavity via a channel. Molecular dynamics analysis implied stable binding of DFA I and inulobiose to the active site with surrounding water molecules. Taken together, these results establish DFA I synthase/hydrolase as a member of a new glycoside hydrolase family (GH172).
Assuntos
Proteínas de Bactérias/química , Bifidobacterium/enzimologia , Glicosídeo Hidrolases/química , Modelos Moleculares , Oligossacarídeos/química , Cristalografia por Raios X , Glicosídeo Hidrolases/classificaçãoRESUMO
ß-l-Arabinofuranosidase HypBA1 from Bifidobacterium longum belongs to the glycoside hydrolase family 127. At the active site of HypBA1, a cysteine residue (Cys417) coordinates with a Zn2+ atom and functions as the catalytic nucleophile for the anomer-retaining hydrolytic reaction. In this study, the role of Zn2+ ion and cysteine in catalysis as well as the substrate-bound structure were studied based on biochemical and crystallographic approaches. The enzymatic activity of HypBA1 decreased after dialysis in the presence of EDTA and guanidine hydrochloride and was then recovered by the addition of Zn2+. The Michaelis complex structure was determined using a crystal of a mutant at the acid/base catalyst residue (E322Q) soaked in a solution containing the substrate p-nitrophenyl-ß-l-arabinofuranoside. To investigate the covalent thioglycosyl enzyme intermediate structure, synthetic inhibitors of l-arabinofuranosyl haloacetamide derivatives with different anomer configurations were used to target the nucleophilic cysteine. In the crystal structure of HypBA1, ß-configured l-arabinofuranosylamide formed a covalent link with Cys417, whereas α-configured l-arabinofuranosylamide was linked to a noncatalytic residue Cys415. Mass spectrometric analysis indicated that Cys415 was also reactive with the probe molecule. With the ß-configured inhibitor, the arabinofuranoside moiety was correctly positioned at the subsite and the active site integrity was retained to successfully mimic the covalent intermediate state.
Assuntos
Cisteína , Zinco , Catálise , Domínio Catalítico , Cristalografia por Raios X , Cisteína/química , Glicosídeo Hidrolases/química , Especificidade por SubstratoRESUMO
In type II polyketide synthases (PKSs), the ketosynthase-chain length factor (KS-CLF) complex catalyzes polyketide chain elongation with the acyl carrier protein (ACP). Highly reducing type II PKSs, represented by IgaPKS, produce polyene structures instead of the well-known aromatic skeletons. Here, we report the crystal structures of the Iga11-Iga12 (KS-CLF) heterodimer and the covalently cross-linked Iga10=Iga11-Iga12 (ACP=KS-CLF) tripartite complex. The latter structure revealed the molecular basis of the interaction between Iga10 and Iga11-Iga12, which differs from that between the ACP and KS of Escherichia coli fatty acid synthase. Furthermore, the reaction pocket structure and site-directed mutagenesis revealed that the negative charge of Asp 113 of Iga11 prevents further condensation using a ß-ketoacyl product as a substrate, which distinguishes IgaPKS from typical type II PKSs. This work will facilitate the future rational design of PKSs.
Assuntos
Proteína de Transporte de Acila/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Ácido Graxo Sintases/química , Policetídeo Sintases/química , Policetídeos/química , Proteína de Transporte de Acila/genética , Proteína de Transporte de Acila/metabolismo , Biocatálise , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces/enzimologia , Streptomyces/genética , Especificidade por SubstratoRESUMO
Pneumococcal cell surface-exposed choline-binding proteins (CBPs) play pivotal roles in multiple infectious processes with pneumococci. Intracellular pneumococci can be recognized at multiple steps during bactericidal autophagy. However, whether CBPs are involved in pneumococci-induced autophagic processes remains unknown. In this study, we demonstrate that CbpC from S. pneumoniae strain TIGR4 activates autophagy through an interaction with Atg14. However, S. pneumoniae also interferes with autophagy by deploying CbpC as a decoy to cause autophagic degradation of Atg14 through an interaction with p62/SQSTM1. Thus, S. pneumoniae suppresses the autophagic degradation of intracellular pneumococci and survives within cells. Domain analysis reveals that the coiled-coil domain of Atg14 and residue Y83 of the dp3 domain in the N-terminal region of CbpC are crucial for both the CbpC-Atg14 interaction and the subsequent autophagic degradation of Atg14. Although homology modeling indicates that CbpC orthologs have similar structures in the dp3 domain, autophagy induction through Atg14 binding is an intrinsic property of CbpC. Our data provide novel insights into the evolutionary hijacking of host-defense systems by intracellular pneumococci.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Proteínas Relacionadas à Autofagia , Autofagia , Proteínas de Bactérias/metabolismo , Streptococcus pneumoniae , Animais , Proteínas Relacionadas à Autofagia/genética , Linhagem Celular , Humanos , Proteínas de Membrana , Camundongos , Streptococcus pneumoniae/genéticaRESUMO
Methyl ß-l-arabinofuranosyl-(1 â 2)-, -(1 â 3)-, and -(1 â 5)-α-l-arabinofuranosides have been stereoselectively synthesized through 2-naphthylmethyl ether-mediated intramolecular aglycon delivery (NAP-IAD), whose ß-linkages were confirmed by NMR analysis on the 3JH1-H2 coupling constant and 13C chemical shift of C1. The NAP-IAD approach was simply extended for the synthesis of trisaccharide motifs possessing ß-l-arabinofuranosyl-(1 â 5)-l-arabinofuranosyl non-reducing terminal structure with the branched ß-l-arabinofuranosyl-(1 â 5)-[α-l-arabinofuranosyl-(1 â 3)]-α-l-arabinofuranosyl and the liner ß-l-arabinofuranosyl-(1 â 5)-ß-l-arabinofuranosyl-(1 â 5)-ß-l-arabinofuranosyl structures in olive arabinan and dinoflagellate polyethers, respectively. The results on the substrate specificity of a bifidobacterial ß-l-arabinofuranosidase HypBA1 using the regioisomers indicated that HypBA1 could hydrolyze all three linkages however behaved clearly less active to ß-(1 â 5)-linked disaccharide than other two regioisomers including the proposed natural degradation product, ß-(1 â 2)-linked one from plant extracellular matrix such as extensin. On the other hand, Xanthomonas XeHypBA1 was found to hydrolyze all three disaccharides as the substrate with higher specificity to ß-(1 â 2)-linkage than bifidobacterial HypBA1.
Assuntos
Dissacarídeos , Glicosídeo Hidrolases , Glicosídeo Hidrolases/metabolismo , Especificidade por SubstratoRESUMO
To understand the precise mechanism of the glycoside hydrolase (GH) family 127, a cysteine ß-l-arabinofuranosidase (Arafase) - HypBA1 - has been isolated from Bifidobacterium longum in the human Gut microbiota, and the design and synthesis of the mechanism-based inhibitors such as l-Araf-haloacetamides have been carried out. The α-l-Araf-azide derivative was used as the monoglycosylamine equivalent to afford the l-Araf-chloroacetamides (α/ß-1-Cl) as well as bromoacetamides (α/ß-1-Br) in highly stereoselective manner through Staudinger reaction followed by amide formation with/without anomerization. Against HypBA1, the probes 1, especially in the case of α/ß-1-Br inhibited the hydrolysis. Conformational implications of these observations are discussed in this manuscript. Additional examinations using l-Araf-azides (α/ß-5) resulted in further mechanistic observations of the GH127/146 cysteine glycosidases, including the hydrolysis of ß-5 as the substrate and oxidative inhibition by α-5 using the GH127 homologue.
RESUMO
Glycoside hydrolase family 136 (GH136) was established after the discovery and structural analysis of lacto-N-biosidase (LNBase) from the infant gut bacterium Bifidobacterium longum subsp. longum JCM1217 (BlLnbX). Homologous genes of BlLnbX are widely distributed in the genomes of human gut bacteria and monkey Bifidobacterium spp., although only 2 crystal structures were reported in the GH136 family. Cell suspensions of Bifidobacterium saguini, Tyzzerella nexilis, and Ruminococcus lactaris exhibited the LNBase activity. Recombinant LNBases of these 3 species were functionally expressed with their specific chaperones in Escherichia coli, and their kinetic parameters against p-nitrophenol substrates were determined. The crystal structures of the LNBases from B. saguini and T. nexilis in complex with lacto-N-biose I were determined at 2.51 and 1.92 Å resolutions, respectively. These structures conserve a ß-helix fold characteristic of GH136 and the catalytic residues, but they lack the metal ions that were present in BlLnbX.
Assuntos
Proteínas de Bactérias , Oligossacarídeos , Animais , Proteínas de Bactérias/química , Glicosídeo Hidrolases/química , Haplorrinos , Humanos , Leite Humano , Oligossacarídeos/químicaRESUMO
Bifidobacteria have attracted significant attention because they provide health-promoting effects in the human gut. In this review, we present a current overview of the three-dimensional structures of bifidobacterial proteins involved in carbohydrate uptake, degradation, and metabolism. As predominant early colonizers of the infant's gut, distinct bifidobacterial species are equipped with a panel of transporters and enzymes specific for human milk oligosaccharides (HMOs). Interestingly, Bifidobacterium bifidum and Bifidobacterium longum possess lacto-N-biosidases with unrelated structural folds to release the disaccharide lacto-N-biose from HMOs, suggesting the convergent evolution of this activity from different ancestral proteins. The crystal structures of enzymes that confer the degradation of glycans from the mucin glycoprotein layer provide a structural basis for the utilization of this sustainable nutrient in the gastrointestinal tract. The utilization of several plant dietary oligosaccharides has been studied in detail, and the prime importance of oligosaccharide-specific ATP-binding cassette (ABC) transporters in glycan utilisations by bifidobacteria has been revealed. The structural elements underpinning the high selectivity and roles of ABC transporter binding proteins in establishing competitive growth on preferred oligosaccharides are discussed. Distinct ABC transporters are conserved across several bifidobacterial species, e.g. those targeting arabinoxylooligosaccharide and α-1,6-galactosides/glucosides. Less prevalent transporters, e.g. targeting ß-mannooligosaccharides, may contribute to the metabolic specialisation within Bifidobacterium. Some bifidobacterial species have established symbiotic relationships with humans. Structural studies of carbohydrate-utilizing systems in Bifidobacterium have revealed the interesting history of molecular coevolution with the host, as highlighted by the early selection of bifidobacteria by mucin and breast milk glycans.
Assuntos
Proteínas de Bactérias/química , Bifidobacterium/enzimologia , Bifidobacterium/metabolismo , Metabolismo dos Carboidratos , Oligossacarídeos/metabolismo , Conformação Proteica , Aldeído Liases/química , Aldeído Liases/metabolismo , Proteínas de Bactérias/metabolismo , Bifidobacterium/classificação , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Oligossacarídeos/química , Oxirredutases/química , Oxirredutases/metabolismo , Especificidade da Espécie , Especificidade por SubstratoRESUMO
The recent discovery of zinc-dependent retaining glycoside hydrolases (GHs), with active sites built around a Zn(Cys)3 (Glu) coordination complex, has presented unresolved mechanistic questions. In particular, the proposed mechanism, depending on a Zn-coordinated cysteine nucleophile and passing through a thioglycosyl enzyme intermediate, remains controversial. This is primarily due to the expected stability of the intermediate C-S bond. To facilitate the study of this atypical mechanism, we report the synthesis of a cyclophellitol-derived ß-l-arabinofuranosidase inhibitor, hypothesised to react with the catalytic nucleophile to form a non-hydrolysable adduct analogous to the mechanistic covalent intermediate. This ß-l-arabinofuranosidase inhibitor reacts exclusively with the proposed cysteine thiol catalytic nucleophiles of representatives of GH families 127 and 146. X-ray crystal structures determined for the resulting adducts enable MD and QM/MM simulations, which provide insight into the mechanism of thioglycosyl enzyme intermediate breakdown. Leveraging the unique chemistry of cyclophellitol derivatives, the structures and simulations presented here support the assignment of a zinc-coordinated cysteine as the catalytic nucleophile and illuminate the finely tuned energetics of this remarkable metalloenzyme clan.
Assuntos
Cicloexanóis/metabolismo , Cisteína/metabolismo , Inibidores Enzimáticos/metabolismo , Glicosídeo Hidrolases/metabolismo , Biocatálise , Cristalografia por Raios X , Cicloexanóis/química , Cicloexanóis/farmacologia , Cisteína/química , Teoria da Densidade Funcional , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/química , Simulação de Dinâmica Molecular , Estrutura MolecularRESUMO
N-Linked glycans play important roles in various cellular and immunological events. Endo-ß-N-acetylglucosaminidase (ENGase) can release or transglycosylate N-glycans and is a promising tool for the chemoenzymatic synthesis of glycoproteins with homogeneously modified glycans. The ability of ENGases to act on core-fucosylated glycans is a key factor determining their therapeutic utility because mammalian N-glycans are frequently α-1,6-fucosylated. Although the biochemistries and structures of various ENGases have been studied extensively, the structural basis for the recognition of the core fucose and the asparagine-linked GlcNAc is unclear. Herein, we determined the crystal structures of a core fucose-specific ENGase from the caterpillar fungus Cordyceps militaris (Endo-CoM), which belongs to glycoside hydrolase family 18. Structures complexed with fucose-containing ligands were determined at 1.75-2.35 Å resolutions. The fucose moiety linked to GlcNAc is extensively recognized by protein residues in a round-shaped pocket, whereas the asparagine moiety linked to the GlcNAc is exposed to the solvent. The N-glycan-binding cleft of Endo-CoM is Y-shaped, and several lysine and arginine residues are present at its terminal regions. These structural features were consistent with the activity of Endo-CoM on fucose-containing glycans on rituximab (IgG) and its preference for a sialobiantennary substrate. Comparisons with other ENGases provided structural insights into their core fucose tolerance and specificity. In particular, Endo-F3, a known core fucose-specific ENGase, has a similar fucose-binding pocket, but the surrounding residues are not shared with Endo-CoM. Our study provides a foothold for protein engineering to develop enzymatic tools for the preparation of more effective therapeutic antibodies.
Assuntos
Acetilglucosaminidase/química , Acetilglucosaminidase/metabolismo , Cordyceps/enzimologia , Fucose/metabolismo , Polissacarídeos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Glicosilação , Modelos Moleculares , Especificidade por SubstratoRESUMO
Cytochrome P450 enzymes (P450 or CYP) are some of the most versatile biocatalysts, and offer advantages for oxidizing unreactive C-H bonds in mild conditions. In this study, we identified a novel cytochrome P450 154C2 from Streptomyces avermitilis and characterized its function in 2α-hydroxylation of testosterone with regio- and stereoselectivity. To investigate the efficiency of electron transfer, we conducted biotransformation using two different P450 redox partners-RhFRED (RhF reductase domain) from Rhodococcus sp. and Pdx (putidaredoxin)/Pdr (putidaredoxin reductase) from Pseudomonas putida and revealed that RhFRED was more effective than Pdx/Pdr, especially in vivo. The Km and kcat values for testosterone were estimated to be 0.16 ± 0.05 mM and 0.13 ± 0.02 min-1, and kcat/Km was 0.81 min-1 mM-1. We also determined the crystal structure of the substrate-free form of CYP154C2 at 1.5 Å resolution. The structure has a closed conformation, and the substrate binding pocket is narrow, which can explain the strict substrate specificity of the enzyme.