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1.
J Biol Chem ; 298(3): 101670, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-35120929

RESUMEN

Xylan is the most common hemicellulose in plant cell walls, though the structure of xylan polymers differs between plant species. Here, to gain a better understanding of fungal xylan degradation systems, which can enhance enzymatic saccharification of plant cell walls in industrial processes, we conducted a comparative study of two glycoside hydrolase family 3 (GH3) ß-xylosidases (Bxls), one from the basidiomycete Phanerochaete chrysosporium (PcBxl3), and the other from the ascomycete Trichoderma reesei (TrXyl3A). A comparison of the crystal structures of the two enzymes, both with saccharide bound at the catalytic center, provided insight into the basis of substrate binding at each subsite. PcBxl3 has a substrate-binding pocket at subsite -1, while TrXyl3A has an extra loop that contains additional binding subsites. Furthermore, kinetic experiments revealed that PcBxl3 degraded xylooligosaccharides faster than TrXyl3A, while the KM values of TrXyl3A were lower than those of PcBxl3. The relationship between substrate specificity and degree of polymerization of substrates suggested that PcBxl3 preferentially degrades xylobiose (X2), while TrXyl3A degrades longer xylooligosaccharides. Moreover, docking simulation supported the existence of extended positive subsites of TrXyl3A in the extra loop located at the N-terminus of the protein. Finally, phylogenetic analysis suggests that wood-decaying basidiomycetes use Bxls such as PcBxl3 that act efficiently on xylan structures from woody plants, whereas molds use instead Bxls that efficiently degrade xylan from grass. Our results provide added insights into fungal efficient xylan degradation systems.


Asunto(s)
Ascomicetos , Phanerochaete , Xilanos , Xilosidasas , Ascomicetos/enzimología , Ascomicetos/genética , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Phanerochaete/enzimología , Phanerochaete/genética , Filogenia , Especificidad por Sustrato , Xilanos/metabolismo , Xilosidasas/química , Xilosidasas/genética , Xilosidasas/metabolismo
2.
J Biol Chem ; 289(45): 31624-37, 2014 Nov 07.
Artículo en Inglés | MEDLINE | ID: mdl-25164811

RESUMEN

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/química
3.
J Biol Chem ; 288(8): 5861-72, 2013 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-23303184

RESUMEN

Root rot fungi of the Heterobasidion annosum complex are the most damaging pathogens in temperate forests, and the recently sequenced Heterobasidion irregulare genome revealed over 280 carbohydrate-active enzymes. Here, H. irregulare was grown on biomass, and the most abundant protein in the culture filtrate was identified as the only family 7 glycoside hydrolase in the genome, which consists of a single catalytic domain, lacking a linker and carbohydrate-binding module. The enzyme, HirCel7A, was characterized biochemically to determine the optimal conditions for activity. HirCel7A was crystallized and the structure, refined at 1.7 Å resolution, confirms that HirCel7A is a cellobiohydrolase rather than an endoglucanase, with a cellulose-binding tunnel that is more closed than Phanerochaete chrysosporium Cel7D and more open than Hypocrea jecorina Cel7A, suggesting intermediate enzyme properties. Molecular simulations were conducted to ascertain differences in enzyme-ligand interactions, ligand solvation, and loop flexibility between the family 7 glycoside hydrolase cellobiohydrolases from H. irregulare, H. jecorina, and P. chrysosporium. The structural comparisons and simulations suggest significant differences in enzyme-ligand interactions at the tunnel entrance in the -7 to -4 binding sites and suggest that a tyrosine residue at the tunnel entrance of HirCel7A may serve as an additional ligand-binding site. Additionally, the loops over the active site in H. jecorina Cel7A are more closed than loops in the other two enzymes, which has implications for the degree of processivity, endo-initiation, and substrate dissociation. Overall, this study highlights molecular level features important to understanding this biologically and industrially important family of glycoside hydrolases.


Asunto(s)
Celulosa 1,4-beta-Celobiosidasa/metabolismo , Glicósido Hidrolasas/fisiología , Phanerochaete/metabolismo , Árboles/microbiología , Trichoderma/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Biocombustibles , Celulasa/química , Celulasa/metabolismo , Celulosa/metabolismo , Simulación por Computador , Cristalografía por Rayos X/métodos , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Hypocrea/metabolismo , Ligandos , Conformación Molecular , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido
4.
Biochem Biophys Res Commun ; 382(2): 430-3, 2009 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-19285960

RESUMEN

Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK) can additionally sensitize human cancer cell lines towards the anti-cancer drug gemcitabine. We show that this property is based on the Dm-dNK ability to efficiently phosphorylate gemcitabine. The 2.2A resolution structure of Dm-dNK in complex with gemcitabine shows that the residues Tyr70 and Arg105 play a crucial role in the firm positioning of gemcitabine by extra interactions made by the fluoride atoms. This explains why gemcitabine is a good substrate for Dm-dNK.


Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Desoxicitidina/análogos & derivados , Drosophila melanogaster/enzimología , Resistencia a Antineoplásicos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Antimetabolitos Antineoplásicos/química , Antimetabolitos Antineoplásicos/metabolismo , Línea Celular Tumoral , Desoxicitidina/química , Desoxicitidina/metabolismo , Desoxicitidina/farmacología , Humanos , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Conformación Proteica , Relación Estructura-Actividad , Especificidad por Sustrato , Gemcitabina
5.
J Mol Biol ; 369(3): 653-64, 2007 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-17448496

RESUMEN

The salvage of deoxyribonucleosides in the social amoeba Dictyostelium discoideum, which has an extremely A+T-rich genome, was investigated. All native deoxyribonucleosides were phosphorylated by D. discoideum cell extracts and we subcloned three deoxyribonucleoside kinase (dNK) encoding genes. D. discoideum thymidine kinase was similar to the human thymidine kinase 1 and was specific for thymidine with a K(m) of 5.1 microM. The other two cloned kinases were phylogenetically closer to bacterial deoxyribonucleoside kinases than to the eukaryotic enzymes. D. discoideum deoxyadenosine kinase (DddAK) had a K(m) for deoxyadenosine of 22.7 microM and a k(cat) of 3.7 s(-1) and could not efficiently phosphorylate any other native deoxyribonucleoside. D. discoideum deoxyguanosine kinase was also a purine-specific kinase and phosphorylated significantly only deoxyguanosine, with a K(m) of 1.4 microM and a k(cat) of 3 s(-1). The two purine-specific deoxyribonucleoside kinases could represent ancient enzymes present in the common ancestor of bacteria and eukaryotes but remaining only in a few eukaryote lineages. The narrow substrate specificity of the D. discoideum dNKs reflects the biased genome composition and we attempted to explain the strict preference of DddAK for deoxyadenosine by modeling the active center with different substrates. Apart from its native substrate, deoxyadenosine, DddAK efficiently phosphorylated fludarabine. Hence, DddAK could be used in the enzymatic production of fludarabine monophosphate, a drug used in the treatment of chronic lymphocytic leukemia.


Asunto(s)
Desoxirribonucleósidos/química , Dictyostelium/metabolismo , Regulación de la Expresión Génica , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Purinas/química , Animales , Antineoplásicos/farmacología , Diferenciación Celular , Dictyostelium/genética , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación , Proteínas Recombinantes/química , Especificidad por Sustrato , Vidarabina/análogos & derivados , Vidarabina/química
6.
Acta Crystallogr F Struct Biol Commun ; 74(Pt 12): 787-796, 2018 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-30511673

RESUMEN

The glycoside hydrolase family 3 (GH3) ß-glucosidases are a structurally diverse family of enzymes. Cel3A from Neurospora crassa (NcCel3A) belongs to a subfamily of key enzymes that are crucial for industrial biomass degradation. ß-Glucosidases hydrolyse the ß-1,4 bond at the nonreducing end of cellodextrins. The hydrolysis of cellobiose is of special importance as its accumulation inhibits other cellulases acting on crystalline cellulose. Here, the crystal structure of the biologically relevant dimeric form of NcCel3A is reported. The structure has been refined to 2.25 Šresolution, with an Rcryst and Rfree of 0.18 and 0.22, respectively. NcCel3A is an extensively N-glycosylated glycoprotein that shares 46% sequence identity with Hypocrea jecorina Cel3A, the structure of which has recently been published, and 61% sequence identity with the thermophilic ß-glucosidase from Rasamsonia emersonii. NcCel3A is a three-domain protein with a number of extended loops that deepen the active-site cleft of the enzyme. These structures characterize this subfamily of GH3 ß-glucosidases and account for the high cellobiose specificity of this subfamily.


Asunto(s)
Glicósido Hidrolasas/química , Neurospora crassa/química , beta-Glucosidasa/química , Cristalización , Glicósido Hidrolasas/biosíntesis , Neurospora crassa/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , beta-Glucosidasa/biosíntesis
7.
Nucleic Acids Res ; 33(21): 6920-30, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-16332695

RESUMEN

Divalent metal ions promote hydrolysis of RNA backbones generating 5'OH and 2';3'P as cleavage products. In these reactions, the neighboring 2'OH act as the nucleophile. RNA catalyzed reactions also require divalent metal ions and a number of different metal ions function in RNA mediated cleavage of RNA. In one case, the LZV leadzyme, it was shown that this catalytic RNA requires lead for catalysis. So far, none of the naturally isolated ribozymes have been demonstrated to use lead to activate the nucleophile. Here we provide evidence that RNase P RNA, a naturally trans-acting ribozyme, has leadzyme properties. But, in contrast to LZV RNA, RNase P RNA mediated cleavage promoted by Pb2+ results in 5' phosphate and 3'OH as cleavage products. Based on our findings, we infer that Pb2+ activates H2O to act as the nucleophile and we identified residues both in the substrate and RNase P RNA that most likely influenced the positioning of Pb2+ at the cleavage site. Our data suggest that Pb2+ can promote cleavage of RNA by activating either an inner sphere H2O or a neighboring 2'OH to act as nucleophile.


Asunto(s)
Plomo/química , ARN Catalítico/química , Ribonucleasa P/química , Secuencia de Bases , Cationes Bivalentes/química , Proteínas de Escherichia coli/metabolismo , Cinética , Magnesio/química , Datos de Secuencia Molecular , Purinas/química , ARN/química , ARN/metabolismo , ARN Catalítico/metabolismo , Ribonucleasa P/metabolismo
8.
Biochemistry ; 42(19): 5706-12, 2003 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-12741827

RESUMEN

Deoxyribonucleoside kinases are feedback inhibited by the final products of the salvage pathway, the deoxyribonucleoside triphosphates. In the present study, the mechanism of feedback inhibition is presented based on the crystal structure of a complex between the fruit fly deoxyribonucleoside kinase and its feedback inhibitor deoxythymidine triphosphate. The inhibitor was found to be bound as a bisubstrate inhibitor with its nucleoside part in the nucleoside binding site and with its phosphate groups partially occupying the phosphate donor site. The overall structure of the enzyme--inhibitor complex is very similar to the enzyme--substrate complexes with deoxythymidine and deoxycytidine, except for a conformational change within a region otherwise directly involved in catalysis. This conformational change involves a magnesium ion, which is coordinated in the inhibitor complex to the phosphates and to the primary base, Glu52, that normally is positioned close to the 5'-OH of the substrate deoxyribose.


Asunto(s)
Desoxirribonucleósidos/metabolismo , Drosophila melanogaster/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Cristalografía por Rayos X , Drosophila melanogaster/genética , Retroalimentación , Cinética , Modelos Moleculares , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Electricidad Estática , Nucleótidos de Timina/metabolismo
9.
Proc Natl Acad Sci U S A ; 101(52): 17970-5, 2004 Dec 28.
Artículo en Inglés | MEDLINE | ID: mdl-15611477

RESUMEN

Cytosolic thymidine kinase 1, TK1, is a well known cell-cycle-regulated enzyme of importance in nucleotide metabolism as well as an activator of antiviral and anticancer drugs such as 3'-azido-3'-deoxythymidine (AZT). We have now determined the structures of the TK1 family, the human and Ureaplasma urealyticum enzymes, in complex with the feedback inhibitor dTTP. The TK1s have a tetrameric structure in which each subunit contains an alpha/beta-domain that is similar to ATPase domains of members of the RecA structural family and a domain containing a structural zinc. The zinc ion connects beta-structures at the root of a beta-ribbon that forms a stem that widens to a lasso-type loop. The thymidine of dTTP is hydrogen-bonded to main-chain atoms predominantly coming from the lasso loop. This binding is in contrast to other deoxyribonucleoside kinases where specific interactions occur with side chains. The TK1 structure differs fundamentally from the structures of the other deoxyribonucleoside kinases, indicating a different evolutionary origin.


Asunto(s)
Mycoplasma/enzimología , Timidina Quinasa/química , Secuencia de Aminoácidos , Antimetabolitos/farmacología , Sitios de Unión , Cristalización , Desoxirribonucleósidos/química , Evolución Molecular , Humanos , Enlace de Hidrógeno , Iones , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Nucleótidos de Timina/química , Ureaplasma urealyticum/enzimología , Zidovudina/farmacología , Zinc/química
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