RESUMEN
Lignin is a combinatorial polymer comprising monoaromatic units that are linked via covalent bonds. Although lignin is a potential source of valuable aromatic chemicals, its recalcitrance to chemical or biological digestion presents major obstacles to both the production of second-generation biofuels and the generation of valuable coproducts from lignin's monoaromatic units. Degradation of lignin has been relatively well characterized in fungi, but it is less well understood in bacteria. A catabolic pathway for the enzymatic breakdown of aromatic oligomers linked via ß-aryl ether bonds typically found in lignin has been reported in the bacterium Sphingobium sp. SYK-6. Here, we present x-ray crystal structures and biochemical characterization of the glutathione-dependent ß-etherases, LigE and LigF, from this pathway. The crystal structures show that both enzymes belong to the canonical two-domain fold and glutathione binding site architecture of the glutathione S-transferase family. Mutagenesis of the conserved active site serine in both LigE and LigF shows that, whereas the enzymatic activity is reduced, this amino acid side chain is not absolutely essential for catalysis. The results include descriptions of cofactor binding sites, substrate binding sites, and catalytic mechanisms. Because ß-aryl ether bonds account for 50-70% of all interunit linkages in lignin, understanding the mechanism of enzymatic ß-aryl ether cleavage has significant potential for informing ongoing studies on the valorization of lignin.
Asunto(s)
Proteínas Bacterianas/química , Dominio Catalítico , Lignina/metabolismo , Oxidorreductasas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia Conservada , Datos de Secuencia Molecular , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Unión Proteica , Proteobacteria/enzimología , Especificidad por SustratoRESUMEN
Cellulase mixtures from Hypocrea jecorina are commonly used for the saccharification of cellulose in biotechnical applications. The most abundant ß-glucosidase in the mesophilic fungus Hypocrea jecorina is HjCel3A, which hydrolyzes the ß-linkage between two adjacent molecules in dimers and short oligomers of glucose. It has been shown that enhanced levels of HjCel3A in H. jecorina cellulase mixtures benefit the conversion of cellulose to glucose. Biochemical characterization of HjCel3A shows that the enzyme efficiently hydrolyzes (1,4)- as well as (1,2)-, (1,3)-, and (1,6)-ß-D-linked disaccharides. For crystallization studies, HjCel3A was produced in both H. jecorina (HjCel3A) and Pichia pastoris (Pp-HjCel3A). Whereas the thermostabilities of HjCel3A and Pp-HjCel3A are the same, Pp-HjCel3A has a higher degree of N-linked glycosylation. Here, we present x-ray structures of HjCel3A with and without glucose bound in the active site. The structures have a three-domain architecture as observed previously for other glycoside hydrolase family 3 ß-glucosidases. Both production hosts resulted in HjCel3A structures that have N-linked glycosylations at Asn(208) and Asn(310). In H. jecorina-produced HjCel3A, a single N-acetylglucosamine is present at both sites, whereas in Pp-HjCel3A, the P. pastoris-produced HjCel3A enzyme, the glycan chains consist of 8 or 4 saccharides. The glycosylations are involved in intermolecular contacts in the structures derived from either host. Due to the different sizes of the glycosylations, the interactions result in different crystal forms for the two protein forms.
Asunto(s)
Proteínas Fúngicas/química , Glucosidasas/química , Hypocrea/enzimología , beta-Glucosidasa/química , Biomasa , Dominio Catalítico , Celulasa/química , Cristalografía por Rayos X , Glucosa/química , Glucósidos/química , Glicosilación , Enlace de Hidrógeno , Hidrólisis , Ligandos , Espectrometría de Masas , Nitrobencenos/química , Oligosacáridos/química , Pichia/metabolismo , Especificidad por Sustrato , Temperatura , Xilosa/análogos & derivados , Xilosa/químicaRESUMEN
Glycosyltransferases are useful synthetic catalysts for generating natural products with sugar moieties. Although several natural product glycosyltransferase structures have been reported, design principles of glycosyltransferase engineering for the generation of glycodiversified natural products has fallen short of its promise, partly due to a lack of understanding of the relationship between structure and function. Here, we report structures of all four calicheamicin glycosyltransferases (CalG1, CalG2, CalG3, and CalG4), whose catalytic functions are clearly regiospecific. Comparison of these four structures reveals a conserved sugar donor binding motif and the principles of acceptor binding region reshaping. Among them, CalG2 possesses a unique catalytic motif for glycosylation of hydroxylamine. Multiple glycosyltransferase structures in a single natural product biosynthetic pathway are a valuable resource for understanding regiospecific reactions and substrate selectivities and will help future glycosyltransferase engineering.
Asunto(s)
Aminoglicósidos/biosíntesis , Antibióticos Antineoplásicos/biosíntesis , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Ingeniería de Proteínas/métodos , Dominios y Motivos de Interacción de Proteínas/genética , Carbohidratos/química , Enediinos/química , Hidroxilaminas/metabolismoRESUMEN
Angiosperms represent most of the terrestrial plants and are the primary research focus for the conversion of biomass to liquid fuels and coproducts. Lignin limits our access to fibers and represents a large fraction of the chemical energy stored in plant cell walls. Recently, the incorporation of monolignol ferulates into lignin polymers was accomplished via the engineering of an exotic transferase into commercially relevant poplar. We report that various angiosperm species might have convergently evolved to natively produce lignins that incorporate monolignol ferulate conjugates. We show that this activity may be accomplished by a BAHD feruloyl-coenzyme A monolignol transferase, OsFMT1 (AT5), in rice and its orthologs in other monocots.
RESUMEN
Sugar aminotransferases (SATs) are an important class of tailoring enzymes that catalyze the 5'-pyridoxal phosphate (PLP)-dependent stereo- and regiospecific installation of an amino group from an amino acid donor (typically L-Glu or L-Gln) to a corresponding ketosugar nucleotide acceptor. Herein we report the strategic structural study of two homologous C4 SATs (Micromonospora echinospora CalS13 and Escherichia coli WecE) that utilize identical substrates but differ in their stereochemistry of aminotransfer. This study reveals for the first time a new mode of SAT sugar nucleotide binding and, in conjunction with previously reported SAT structural studies, provides the basis from which to propose a universal model for SAT stereo- and regiochemical control of amine installation. Specifically, the universal model put forth highlights catalytic divergence to derive solely from distinctions within nucleotide sugar orientation upon binding within a relatively fixed SAT active site where the available ligand bound structures of the three out of four representative C3 and C4 SAT examples provide a basis for the overall model. Importantly, this study presents a new predictive model to support SAT functional annotation, biochemical study and rational engineering.
Asunto(s)
Aminas/metabolismo , Aminoácidos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Micromonospora/enzimología , Nucleótidos/metabolismo , Transaminasas/metabolismo , Aminas/química , Aminoácidos/química , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Micromonospora/química , Micromonospora/metabolismo , Modelos Moleculares , Nucleótidos/química , Conformación Proteica , Fosfato de Piridoxal/metabolismo , Especificidad por Sustrato , Transaminasas/químicaRESUMEN
UNLABELLED: Methionine adenosyltransferase (MAT) is a family of enzymes that utilizes ATP and methionine to produce S-adenosylmethionine (AdoMet), the most crucial methyl donor in the biological methylation of biomolecules and bioactive natural products. Here, we report that the MAT from Sulfolobus solfataricus (sMAT), an enzyme from a poorly explored class of the MAT family, has the ability to produce a range of differentially alkylated AdoMet analogs in the presence of non-native methionine analogs and ATP. To investigate the molecular basis for AdoMet analog production, we have crystallized the sMAT in the AdoMet bound, S-adenosylethionine (AdoEth) bound and unbound forms. Notably, among these structures, the AdoEth bound form offers the first MAT structure containing a non-native product, and cumulatively these structures add new structural insight into the MAT family and allow for detailed active site comparison with its homologs in Escherichia coli and human. As a thermostable MAT structure from archaea, the structures herein also provide a basis for future engineering to potentially broaden AdoMet analog production as reagents for methyltransferase-catalyzed 'alkylrandomization' and/or the study of methylation in the context of biological processes. DATABASES: PDB IDs: 4HPV, 4L7I, 4K0B and 4L2Z. EC 2.5.1.6 STRUCTURED DIGITAL ABSTRACT: ⢠sMAT and sMAT bind by x-ray crystallography (View interaction).
Asunto(s)
Proteínas Arqueales/química , Metionina Adenosiltransferasa/química , Sulfolobus solfataricus/enzimología , Secuencias de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Cinética , Metionina/química , Modelos Moleculares , Unión Proteica , Estructura Cuaternaria de Proteína , Especificidad por SustratoRESUMEN
Sugar methyltransferases (MTs) are an important class of tailoring enzymes that catalyze the transfer of a methyl group from S-adenosyl-l-methionine to sugar-based N-, C- and O-nucleophiles. While sugar N- and C-MTs involved in natural product biosynthesis have been found to act on sugar nucleotide substrates prior to a subsequent glycosyltransferase reaction, corresponding sugar O-methylation reactions studied thus far occur after the glycosyltransfer reaction. Herein we report the first in vitro characterization using (1)H-(13)C-gHSQC with isotopically labeled substrates and the X-ray structure determination at 1.55 Å resolution of the TDP-3'-O-rhamnose-methyltransferase CalS11 from Micromonospora echinospora. This study highlights a unique NMR-based methyltransferase assay, implicates CalS11 to be a metal- and general acid/base-dependent O-methyltransferase, and as a first crystal structure for a TDP-hexose-O-methyltransferase, presents a new template for mechanistic studies and/or engineering.