RESUMEN

Glycoside hydrolases (GHs) are pivotal in the hydrolysis of the glycosidic bonds of sugars, which are the main carbon and energy sources. The genome of Marinomonas sp. ef1, an Antarctic bacterium, contains three GHs belonging to family 3. These enzymes have distinct architectures and low sequence identity, suggesting that they originated from separate horizontal gene transfer events. M-GH3_A and M-GH3_B, were found to differ in cold adaptation and substrate specificity. M-GH3_A is a bona fide cold-active enzyme since it retains 20 % activity at 10 °C and exhibits poor long-term thermal stability. On the other hand, M-GH3_B shows mesophilic traits with very low activity at 10 °C (< 5 %) and higher long-term thermal stability. Substrate specificity assays highlight that M-GH3_A is a promiscuous ß-glucosidase mainly active on cellobiose and cellotetraose, whereas M-GH3_B is a ß-xylosidase active on xylan and arabinoxylan. Structural analysis suggests that such functional differences are due to their differently shaped active sites. The active site of M-GH3_A is wider but has a narrower entrance compared to that of M-GH3_B. Genome-based prediction of metabolic pathways suggests that Marinomonas sp. ef1 can use monosaccharides derived from the GH3-catalyzed hydrolysis of oligosaccharides either as a carbon source or for producing osmolytes.

Asunto(s)

Evolución Molecular , Glicósido Hidrolasas , Oligosacáridos , Glicósido Hidrolasas/metabolismo , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/química , Especificidad por Sustrato , Oligosacáridos/metabolismo , Regiones Antárticas , Polisacáridos/metabolismo , Polisacáridos/química , Filogenia , Marinomonas/enzimología , Marinomonas/genética , Organismos Acuáticos/enzimología , Estabilidad de Enzimas , Dominio Catalítico , Hidrólisis

RESUMEN

Cold-active enzymes support life at low temperatures due to their ability to maintain high activity in the cold and can be useful in several biotechnological applications. Although information on the mechanisms of enzyme cold adaptation is still too limited to devise general rules, it appears that very diverse structural and functional changes are exploited in different protein families and within the same family. In this context, we studied the cold adaptation mechanism and the functional properties of a member of the glycoside hydrolase family 1 (GH1) from the Antarctic bacterium Marinomonas sp. ef1. This enzyme exhibits all typical functional hallmarks of cold adaptation, including high catalytic activity at 5 °C, broad substrate specificity, low thermal stability, and higher lability of the active site compared to the overall structure. Analysis of the here-reported crystal structure (1.8 Å resolution) and molecular dynamics simulations suggest that cold activity and thermolability may be due to a flexible region around the active site (residues 298-331), whereas the dynamic behavior of loops flanking the active site (residues 47-61 and 407-413) may favor enzyme-substrate interactions at the optimal temperature of catalysis (Topt) by tethering together protein regions lining the active site. Stapling of the N-terminus onto the surface of the ß-barrel is suggested to partly counterbalance protein flexibility, thus providing a stabilizing effect. The tolerance of the enzyme to glucose and galactose is accounted for by the presence of a "gatekeeping" hydrophobic residue (Leu178), located at the entrance of the active site.

Asunto(s)

Dominio Catalítico , Frío , Glucosa , Glicósido Hidrolasas , Marinomonas , Simulación de Dinámica Molecular , Marinomonas/enzimología , Marinomonas/genética , Marinomonas/química , Especificidad por Sustrato , Glucosa/metabolismo , Cristalografía por Rayos X , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Glicósido Hidrolasas/genética , Regiones Antárticas , Estabilidad de Enzimas , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Conformación Proteica , Secuencia de Aminoácidos

RESUMEN

To survive in cold environments, psychrophilic organisms produce enzymes endowed with high specific activity at low temperature. The structure of these enzymes is usually flexible and mostly thermolabile. In this work, we investigate the structural basis of cold adaptation of a GH42 ß-galactosidase from the psychrophilic Marinomonas ef1. This enzyme couples cold activity with astonishing robustness for a psychrophilic protein, for it retains 23% of its highest activity at 5 °C and it is stable for several days at 37 °C and even 50 °C. Phylogenetic analyses indicate a close relationship with thermophilic ß-galactosidases, suggesting that the present-day enzyme evolved from a thermostable scaffold modeled by environmental selective pressure. The crystallographic structure reveals the overall similarity with GH42 enzymes, along with a hexameric arrangement (dimer of trimers) not found in psychrophilic, mesophilic, and thermophilic homologues. In the quaternary structure, protomers form a large central cavity, whose accessibility to the substrate is promoted by the dynamic behavior of surface loops, even at low temperature. A peculiar cooperative behavior of the enzyme is likely related to the increase of the internal cavity permeability triggered by heating. Overall, our results highlight a novel strategy of enzyme cold adaptation, based on the oligomerization state of the enzyme, which effectively challenges the paradigm of cold activity coupled with intrinsic thermolability. DATABASE: Structural data are available in the Protein Data Bank database under the accession number 6Y2K.

Asunto(s)

Proteínas Bacterianas/química , Galactosa/química , Marinomonas/química , beta-Galactosidasa/química , Secuencia de Aminoácidos , Regiones Antárticas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Frío , Cristalografía por Rayos X , Estabilidad de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Galactosa/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Cinética , Marinomonas/enzimología , Modelos Moleculares , Filogenia , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Termodinámica , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo

RESUMEN

d-Mannose isomerase (MI) reversibly isomerizes d-mannose to d-fructose, and is attractive for producing d-mannose from inexpensive d-fructose. It belongs to the N-acylglucosamine 2-epimerase (AGE) superfamily along with AGE, cellobiose 2-epimerase (CE), and aldose-ketose isomerase (AKI). In this study, Marinomonas mediterranea Marme_2490, showing low sequence identity with any known enzymes, was found to isomerize d-mannose as its primary substrate. Marme_2490 also isomerized d-lyxose and 4-OH d-mannose derivatives (d-talose and 4-O-monosaccharyl-d-mannose). Its activity for d-lyxose is known in other d-mannose isomerizing enzymes, such as MI and AKI, but we identified, for the first time, its activity for 4-OH d-mannose derivatives. Marme_2490 did not isomerize d-glucose, as known MIs do not, while AKI isomerizes both d-mannose and d-glucose. Thus, Marme_2490 was concluded to be an MI. The initial and equilibrium reaction products were analyzed by NMR to illuminate mechanistic information regarding the Marme_2490 reaction. The analysis of the initial reaction product revealed that ß-d-mannose was formed. In the analysis of the equilibrated reaction products in D2O, signals of 2-H of d-mannose and 1-H of d-fructose were clearly detected. This indicates that these protons are not substituted with deuterium from D2O and Marme_2490 catalyzes the intramolecular proton transfer between 1-C and 2-C. The crystal structure of Marme_2490 in a ligand-free form was determined and found that Marme_2490 is formed by an (α/α)6-barrel, which is commonly observed in AGE superfamily enzymes. Despite diverse reaction specificities, the orientations of residues involved in catalysis and substrate binding by Marme_2490 were similar to those in both AKI (Salmonella enterica AKI) and epimerase (Rhodothermus marinus CE). The Marme_2490 structure suggested that the α7→α8 and α11→α12 loops of the catalytic domain participated in the formation of an open substrate-binding site to provide sufficient space to bind 4-OH d-mannose derivatives.

Asunto(s)

Isomerasas Aldosa-Cetosa/química , Isomerasas Aldosa-Cetosa/metabolismo , Marinomonas/enzimología , Disacáridos/química , Disacáridos/metabolismo , Evolución Molecular , Concentración de Iones de Hidrógeno , Isomerismo , Cinética , Filogenia , Especificidad por Sustrato , Temperatura

RESUMEN

The first posttranslational modification step in the biosynthesis of the tryptophan-derived quinone cofactors is the autocatalytic hydroxylation of a specific Trp residue at position C-7 on the indole side chain. Subsequent modifications are catalyzed by modifying enzymes, but the mechanism by which this first step occurs is unknown. LodA possesses a cysteine tryptophylquinone (CTQ) cofactor. Metal analysis as well as spectroscopic and kinetic studies of the mature and precursor forms of a D512A LodA variant provides evidence that copper is required for the initial hydroxylation of the precursor protein and that if alternative metals are bound, the modification does not occur and the precursor is unstable. It is shown that the mature native LodA also contains loosely bound copper, which affects the visible absorbance spectrum and quenches the fluorescence spectrum that is attributed to the mature CTQ cofactor. When copper is removed, the fluorescence appears, and when it is added back to the protein, the fluorescence is quenched, indicating that copper reversibly binds in the proximity of CTQ. Removal of copper does not diminish the enzymatic activity of LodA. This distinguishes LodA from enzymes with protein-derived tyrosylquinone cofactors in which copper is present near the cofactor and is absolutely required for activity. Mechanisms are proposed for the role of copper in the hydroxylation of the unactivated Trp side chain. These results demonstrate that the reason that the highly conserved Asp512 is critical for LodA, and possibly all tryptophylquinone enzymes, is not because it is required for catalysis but because it is necessary for CTQ biosynthesis, more specifically to facilitate the initial copper-dependent hydroxylation of a specific Trp residue.

Asunto(s)

Aminoácido Oxidorreductasas/química , Ácido Aspártico/metabolismo , Cobre/metabolismo , Dipéptidos/metabolismo , Indolquinonas/metabolismo , Triptófano/metabolismo , Aminoácido Oxidorreductasas/metabolismo , Ácido Aspártico/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cobre/química , Dipéptidos/química , Hidroxilación , Indolquinonas/química , Marinomonas/enzimología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Triptófano/química

RESUMEN

Agar and sulfated galactans were isolated from the red seaweeds Gracilariopsis lemaneiformis and Gelidium amansii. A previously purified arylsulfatase from Marinomonas sp. FW-1 was used to remove sulfate groups in agar and sulfated galactans. After enzymatic desulfation, the sulfate content decreased to about 0.16% and gel strength increased about two folds. Moreover, there was no difference between the DNA electrophoresis spectrum on the gel of the arylsulfatase-treated agar and that of the commercial agarose. In order to reveal the desulfation ratio and site, chemical and structural identification of sulfated galactan were carried out. G. amansii sulfated galactan with 7.4% sulfated content was composed of galactose and 3,6-anhydro-l-galactose. Meanwhile, G. lemaneiformis sulfated galactan with 8.5% sulfated content was composed of galactose, 3,6-anhydro-l-galactose, 2-O-methyl-3,6-anhydro-l-galactose and xylose. Data from 13C NMR, FT-IR, GC-MS provided evidence of sulfate groups at C-4 and C-6 of d-galactose and C-6 of l-galactose both in GRAP and GEAP. Data from GC-MS revealed that desulfation was carried out by the arylsulfatase at the sulfate bonds at C-4 and C-6 of d-galactose and C-6 of l-galactose, with a desulfation ratio of 83.4% and 86.0% against GEAP and GRAP, respectively.

Asunto(s)

Agar/química , Arilsulfatasas/metabolismo , Gracilaria/química , Marinomonas/enzimología , Agar/metabolismo , Galactosa/química , Metilación

RESUMEN

Polyphenol oxidase from the marine bacterium Marinomonas mediterranea (MmPPOA) is a membrane-bound, blue, multi-copper laccase of 695 residues. It possesses peculiar properties that distinguish it from known laccases, such as a broad substrate specificity (common to tyrosinases) and a high redox potential. In order to push the biotechnological application of this laccase, the full-length enzyme was overexpressed in Escherichia coli cells with and without a C-terminal His-tag. The previous form, named rMmPPOA-695-His, was purified to homogeneity by HiTrap chelating chromatography following solubilization by 1% SDS in the lysis buffer with an overall yield of ≈1 mg/L fermentation broth and a specific activity of 1.34 U/mg protein on 2,6-dimethoxyphenol as substrate. A truncated enzyme form lacking 58 residues at the N-terminus encompassing the putative membrane binding region, namely rMmPPOA-637-His, was successfully expressed in E. coli as soluble protein and was purified by using the same procedure set-up as for the full-length enzyme. Elimination of the N-terminal sequence decreased the specific activity 15-fold (which was partially restored in the presence of 1 M NaCl) and altered the secondary and tertiary structures and the pH dependence of optimal stability. The recombinant rMmPPOA-695-His showed kinetic properties on catechol higher than for known laccases, a very high thermal stability, and a strong resistance to NaCl, DMSO, and Tween-80, all properties that are required for specific, targeted industrial applications.

Asunto(s)

Clonación Molecular , Lacasa/metabolismo , Marinomonas/enzimología , Catecol Oxidasa/química , Catecol Oxidasa/genética , Catecol Oxidasa/metabolismo , Catecoles/metabolismo , Estabilidad de Enzimas , Escherichia coli/genética , Cinética , Lacasa/química , Lacasa/genética , Lignina/metabolismo , Marinomonas/química , Marinomonas/genética , Marinomonas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidad , Especificidad por Sustrato , Temperatura

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Marinomonas/enzimología , Proteínas Mutantes Quiméricas/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , ARN/metabolismo

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Marinomonas/enzimología , Proteínas Mutantes Quiméricas/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , ARN/metabolismo

RESUMEN

CRISPR systems mediate adaptive immunity in diverse prokaryotes. CRISPR-associated Cas1 and Cas2 proteins have been shown to enable adaptation to new threats in type I and II CRISPR systems by the acquisition of short segments of DNA (spacers) from invasive elements. In several type III CRISPR systems, Cas1 is naturally fused to a reverse transcriptase (RT). In the marine bacterium Marinomonas mediterranea (MMB-1), we showed that a RT-Cas1 fusion protein enables the acquisition of RNA spacers in vivo in a RT-dependent manner. In vitro, the MMB-1 RT-Cas1 and Cas2 proteins catalyze the ligation of RNA segments into the CRISPR array, which is followed by reverse transcription. These observations outline a host-mediated mechanism for reverse information flow from RNA to DNA.

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Marinomonas/enzimología , Proteínas Mutantes Quiméricas/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , ARN/metabolismo , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Secuencia de Bases , Proteínas Asociadas a CRISPR/clasificación , Proteínas Asociadas a CRISPR/genética , ADN/genética , Intrones/genética , Marinomonas/genética , Datos de Secuencia Molecular , Proteínas Mutantes Quiméricas/clasificación , Proteínas Mutantes Quiméricas/genética , Filogenia , Estructura Terciaria de Proteína , ARN/genética , Empalme del ARN , ADN Polimerasa Dirigida por ARN/clasificación , ADN Polimerasa Dirigida por ARN/genética

RESUMEN

A serine protease-producing marine bacterial strain named as PT-1 was isolated and identified as a family of Marinomonas arctica, based on molecular characterization of 16S rRNA gene sequence, phylogenetic tree, and fatty acid composition analyses. Optimized culture conditions for growth of the bacterium PT-1 and production of protease (ProA) were determined to be pH 8.0 in the presence of 5 % NaCl, at 37 °C during 24 h of incubation in the presence of 1.0 % skim milk. The molecular weight of the purified ProA was estimated to be 63-kDa as a major band by SDS-PAGE. We were intrigued to find that the activity of ProA was not inhibited by pepstatin A, chymostatin, and leupeptin known as inhibitors for cysteine protease. However, phenylmethylsulfonyl fluoride (PMSF) completely inhibited protease activity, suggesting that the ProA is like a serine protease. To the best of our knowledge, this is the first report on serine protease of Marinomonas species.

Asunto(s)

Organismos Acuáticos/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Marinomonas/enzimología , Serina Proteasas/química , Serina Proteasas/aislamiento & purificación , Organismos Acuáticos/genética , Proteínas Bacterianas/genética , Marinomonas/genética , Serina Proteasas/genética

RESUMEN

A bacterial strain capable of hydrolyzing sulfate ester bonds of p-nitrophenyl sulfate (pNPS) and agar was isolated from the coast area of Qingdao, China. It was identified as Marinomonas based on its 16S rRNA gene sequence and named as Marinomonas sp. FW-1. An arylsulfatase with a recovery of 13 % and a fold of 12 was purified to a homogeneity using ion exchange and gel filtration chromatographies. The enzyme was composed of a single polypeptide chain with the molecular mass of 33 kDa estimated using SDS-PAGE. The optimal pH and temperature of arylsulfatase were pH 9.0 and 45, respectively. Arylsulfatase was stable over pH 8-11 and at temperature below 55 °C. The K m and V max of this enzyme for the hydrolysis of pNPS were determined to be 13.73 and 270.27 µM/min, respectively. The desulfation ratio against agar from red seaweed Gelidium amansii and Gracilaria lemaneiformis were 86.11 and 89.61 %, respectively. There was no difference between the DNA electrophoresis spectrum on the gel of the arylsulfatase-treated G. amansii agar and that of the commercial agarose. Therefore, this novel alkaline arylsulfatase might have a great potential for application in enzymatic conversion of agar to agarose.

Asunto(s)

Agar/química , Agar/metabolismo , Arilsulfatasas/metabolismo , Marinomonas/enzimología , Algas Marinas/química , Arilsulfatasas/química , Arilsulfatasas/aislamiento & purificación , China , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Hidrólisis , Marinomonas/clasificación , Marinomonas/genética , Marinomonas/aislamiento & purificación , Peso Molecular , Nitrobencenos/metabolismo , ARN Ribosómico 16S/genética , Sefarosa/química , Sefarosa/metabolismo , Temperatura

RESUMEN

For the heterologous production of l-lysine ε-oxidase (LodA), we constructed a new plasmid carrying LodA gene fused in-frame with an antibiotic (phleomycine) resistant gene. The new plasmid was randomly mutated and the mutated plasmids were transformed into Escherichia coli BL21 (DE3) harboring lodB, which encodes a protein (LodB) acting in posttranslational modification of LodA, and active mutants were selected by phleomycin resistance and oxidase activities. One soluble LodA variant isolated by this method contained six silent mutations and one missense mutation. At these mutation points, the codon adaptations at Lys92, Ala550, and Thr646, and the amino acid substitution at His286 to Arg contributed to the production of its functional form. The active form of LodA variant was induced by post-modification of LodB in the heterologous coexpression, and the activity increased with additional NaCl and heat treatment. This is the first report of heterologous production of LodA by random mutagenesis.

Asunto(s)

Aminoácido Oxidorreductasas/genética , Dipéptidos/biosíntesis , Escherichia coli/genética , Indolquinonas/biosíntesis , Marinomonas/enzimología , Aminoácido Oxidorreductasas/química , Aminoácido Oxidorreductasas/metabolismo , Codón , Evolución Molecular Dirigida , Farmacorresistencia Bacteriana/genética , Regulación Enzimológica de la Expresión Génica , Lisina/metabolismo , Marinomonas/genética , Mutagénesis Sitio-Dirigida , Plásmidos , Procesamiento Proteico-Postraduccional/genética

RESUMEN

The lysine-ε-oxidase, LodA, and glycine oxidase, GoxA, from Marinomonas mediteranea each possesses a cysteine tryptophylquinone (CTQ) cofactor. This cofactor is derived from posttranslational modifications which are covalent crosslinking of tryptophan and cysteine residues and incorporation of two oxygen atoms into the indole ring of Trp. In this manuscript, it is shown that the recombinant synthesis of LodA and GoxA containing a fully synthesized CTQ cofactor requires coexpression of a partner flavoprotein, LodB for LodA and GoxB for GoxA, which are not interchangeable. An inactive precursor of LodA or GoxA which contained a monohydroxylated Trp residue and no crosslink to the Cys was isolated from the soluble fraction when they were expressed alone. The structure of LodA revealed an Asp residue close to the cofactor which is conserved in quinohemoprotein amine dehydrogenase (QHNDH), containing CTQ, and methylamine dehydrogenase (MADH) containing tryptophan tryptophylquinone (TTQ) as cofactor. To study the role of this residue in the synthesis of the LodA precursor, Asp-512 was mutated to Ala. When the mutant protein was coexpressed with LodB an inactive protein was isolated which was soluble and contained no modifications at all, suggesting a role for this Asp in the initial LodB-independent hydroxylation of Trp. A similar role had been proposed for this conserved Asp residue in MADH. It is noteworthy that the formation of TTQ in MADH from the precursor also requires an accessory enzyme for its biosynthesis but it is a diheme enzyme MauG and not a flavoprotein. The results presented reveal novel mechanisms of post-translational modification involved in the generation of protein-derived cofactors. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

Asunto(s)

Aminoácido Oxidorreductasas/biosíntesis , Coenzimas/química , Dipéptidos/química , Indolquinonas/química , Marinomonas/enzimología , Proteínas Recombinantes/biosíntesis , Aminoácido Oxidorreductasas/química , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional

RESUMEN

Dimethyl sulfide (DMS) is produced in oceans in vast amounts (>10(7) tons/year) and mediates a wide range of processes from regulating marine life forms to cloud formation. Nonetheless, none of the enzymes that produce DMS from dimethylsulfoniopropionate (DMSP) has been adequately characterized. We describe the expression and purification of DddD from the marine bacterium Marinomonas sp. MWYL1 and its biochemical characterization. We identified DMSP and acetyl-coenzyme A to be DddD's native substrates and Asp602 as the active site residue mediating the CoA-transferase prior to lyase activity. These findings shed light on the biochemical utilization of DMSP in the marine environment.

Asunto(s)

Proteínas Bacterianas/metabolismo , Marinomonas/enzimología , Agua de Mar , Sulfuros/metabolismo

RESUMEN

LodA is a novel lysine-ε-oxidase which possesses a cysteine tryptophylquinone cofactor. It is the first tryptophylquinone enzyme known to function as an oxidase. A steady-state kinetic analysis shows that LodA obeys a ping-pong kinetic mechanism with values of kcat of 0.22±0.04 s(-1), Klysine of 3.2±0.5 µM and KO2 of 37.2±6.1 µM. The kcat exhibited a pH optimum at 7.5 while kcat/Klysine peaked at 7.0 and remained constant to pH 8.5. Alternative electron acceptors could not effectively substitute for O2 in the reaction. A mechanism for the reductive half reaction of LodA is proposed that is consistent with the ping-pong kinetics.

Asunto(s)

Proteínas Bacterianas/química , Dipéptidos/química , Indolquinonas/química , Marinomonas/enzimología , Proteínas/química , Ácido 2-Aminoadípico/análogos & derivados , Ácido 2-Aminoadípico/química , Coenzimas/química , Concentración de Iones de Hidrógeno , Cinética , Lisina/química , Modelos Químicos

RESUMEN

Marinomonas mediterranea is a marine gamma-proteobacterium that synthesizes LodA, a novel L-lysine-ε-oxidase (E.C. 1.4.3.20). This enzyme oxidizes L-lysine generating 2-aminoadipate 6-semialdehyde, ammonium, and hydrogen peroxide. Unlike other L-amino acid oxidases, LodA is not a flavoprotein but contains a quinone cofactor. LodA is encoded by an operon with two genes, lodA and lodB. In the native system, LodB is required for the synthesis of a functional LodA. In this study, we report the recombinant expression of LodA in Escherichia coli using vectors that allow its expression and accumulation in the cytoplasm. To reveal the L-lysine-ε-oxidase activity using the Amplex Red method for hydrogen peroxide detection, it is necessary to first remove the E. coli cytoplasmic catalases. The flavoprotein LodB is the only M. mediterranea protein required in the recombinant system for the generation of the cofactor of LodA. In the absence of LodB, LodA does not contain the quinone cofactor and remains in an inactive form. The results presented indicate that LodB participates in the posttranslational modification of LodA that generates the quinone cofactor.

Asunto(s)

Aminoácido Oxidorreductasas/biosíntesis , Proteínas Bacterianas/metabolismo , Marinomonas/enzimología , Marinomonas/metabolismo , Aminoácido Oxidorreductasas/genética , Proteínas Bacterianas/genética , Coenzimas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Quinonas/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética

RESUMEN

We have determined the x-ray crystal structure of L-lysine ε-oxidase from Marinomonas mediterranea in its native and L-lysine-complex forms at 1.94- and 1.99-Šresolution, respectively. In the native enzyme, electron densities clearly indicate the presence of cysteine tryptophylquinone (CTQ) previously identified in quinohemoprotein amine dehydrogenase. In the L-lysine-complex, an electron density corresponding to the bound L-lysine shows that its ε-amino group is attached to the C6 carbonyl group of CTQ, suggesting the formation of a Schiff-base intermediate. Collectively, the present crystal structure provides the first example of an enzyme employing a tryptophylquinone cofactor in an amine oxidase.

Asunto(s)

Aminoácido Oxidorreductasas/química , Proteínas Bacterianas/química , Coenzimas/química , Dipéptidos/química , Electrones , Indolquinonas/química , Marinomonas/química , Dominio Catalítico , Cristalografía por Rayos X , Cinética , Marinomonas/enzimología , Modelos Moleculares , Bases de Schiff/química

RESUMEN

A novel enzyme with lysine-epsilon oxidase activity was previously described in the marine bacterium Marinomonas mediterranea. This enzyme differs from other l-amino acid oxidases in not being a flavoprotein but containing a quinone cofactor. It is encoded by an operon with two genes lodA and lodB. The first one codes for the oxidase, while the second one encodes a protein required for the expression of the former. Genome sequencing of M. mediterranea has revealed that it contains two additional operons encoding proteins with sequence similarity to LodA. In this study, it is shown that the product of one of such genes, Marme_1655, encodes a protein with glycine oxidase activity. This activity shows important differences in terms of substrate range and sensitivity to inhibitors to other glycine oxidases previously described which are flavoproteins synthesized by Bacillus. The results presented in this study indicate that the products of the genes with different degrees of similarity to lodA detected in bacterial genomes could constitute a reservoir of different oxidases.

Asunto(s)

Aminoácido Oxidorreductasas/aislamiento & purificación , Aminoácido Oxidorreductasas/metabolismo , Marinomonas/enzimología , Aminoácido Oxidorreductasas/genética , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos/metabolismo , Eliminación de Gen , Orden Génico , Prueba de Complementación Genética , Marinomonas/genética , Espectrometría de Masas , Datos de Secuencia Molecular , Especificidad por Sustrato , Espectrometría de Masas en Tándem

RESUMEN

The gene encoding a cold-active and xylose-stimulated ß-glucosidase of Marinomonas MWYL1 was synthesized and expressed in Escherichia coli. The recombinant enzyme (reBglM1) was purified and characterized. The molecular mass of the purified reBglM1 determined by SDS-PAGE agree with the calculated values (50.6 Da). Optima of temperature and pH for enzyme activity were 40 °C and 7.0, respectively. The enzyme exhibited about 20% activity at 5 °C and was stable over the range of pH 5.5-10.0. The presence of xylose significantly enhanced enzyme activity even at higher concentrations up to 600 mM, with maximal stimulatory effect (about 1.45-fold) around 300 mM. The enzyme is active with both glucosides and galactosides and showed high catalytic efficiency (k (cat) = 500.5 s(-1)) for oNPGlc. These characterizations enable the enzyme to be a promising candidate for industrial applications.

Asunto(s)

Marinomonas/enzimología , Proteínas Recombinantes/metabolismo , beta-Glucosidasa/genética , beta-Glucosidasa/metabolismo , Secuencia de Bases , Frío , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Análisis de Secuencia de ADN , Especificidad por Sustrato , Xilosa/metabolismo