RESUMO
Many enzymes can self-assemble into higher-order structures with helical symmetry. A particularly noteworthy example is that of nitrilases, enzymes in which oligomerization of dimers into spiral homo-oligomers is a requirement for their enzymatic function. Nitrilases are widespread in nature where they catalyze the hydrolysis of nitriles into the corresponding carboxylic acid and ammonia. Here, we present the Cryo-EM structure, at 3 Å resolution, of a C-terminal truncate nitrilase from Rhodococcus sp. V51B that assembles in helical filaments. The model comprises a complete turn of the helical arrangement with a substrate-intermediate bound to the catalytic cysteine. The structure was solved having added the substrate to the protein. The length and stability of filaments was made more substantial in the presence of the aromatic substrate, benzonitrile, but not for aliphatic nitriles or dinitriles. The overall structure maintains the topology of the nitrilase family, and the filament is formed by the association of dimers in a chain-like mechanism that stabilizes the spiral. The active site is completely buried inside each monomer, while the substrate binding pocket was observed within the oligomerization interfaces. The present structure is in a closed configuration, judging by the position of the lid, suggesting that the intermediate is one of the covalent adducts. The proximity of the active site to the dimerization and oligomerization interfaces, allows the dimer to sense structural changes once the benzonitrile was bound, and translated to the rest of the filament, stabilizing the helical structure.
Assuntos
Aminoidrolases , Microscopia Crioeletrônica , Nitrilas , Multimerização Proteica , Rhodococcus , Aminoidrolases/química , Aminoidrolases/metabolismo , Aminoidrolases/ultraestrutura , Microscopia Crioeletrônica/métodos , Rhodococcus/enzimologia , Nitrilas/química , Nitrilas/metabolismo , Especificidade por Substrato , Modelos Moleculares , Domínio Catalítico , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , CatáliseRESUMO
The bacterial genus Rhodococcus comprises organisms performing oleaginous behaviors under certain growth conditions and ratios of carbon and nitrogen availability. Rhodococci are outstanding producers of biofuel precursors, where lipid and glycogen metabolisms are closely related. Thus, a better understanding of rhodococcal carbon partitioning requires identifying catalytic steps redirecting sugar moieties to storage molecules. Here, we analyzed two GT4 glycosyl-transferases from Rhodococcus jostii (RjoGlgAb and RjoGlgAc) annotated as α-glucan-α-1,4-glucosyl transferases, putatively involved in glycogen synthesis. Both enzymes were produced in Escherichia coli cells, purified to homogeneity, and kinetically characterized. RjoGlgAb and RjoGlgAc presented the "canonical" glycogen synthase activity and were actives as maltose-1P synthases, although to a different extent. Then, RjoGlgAc is a homologous enzyme to the mycobacterial GlgM, with similar kinetic behavior and glucosyl-donor preference. RjoGlgAc was two orders of magnitude more efficient to glucosylate glucose-1P than glycogen, also using glucosamine-1P as a catalytically efficient aglycon. Instead, RjoGlgAb exhibited both activities with similar kinetic efficiency and preference for short-branched α-1,4-glucans. Curiously, RjoGlgAb presented a super-oligomeric conformation (higher than 15 subunits), representing a novel enzyme with a unique structure-to-function relationship. Kinetic results presented herein constitute a hint to infer on polysaccharides biosynthesis in rhodococci from an enzymological point of view.
Assuntos
Glicosiltransferases , Rhodococcus , Rhodococcus/enzimologia , Rhodococcus/metabolismo , Glicosiltransferases/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/química , Polissacarídeos/metabolismo , Polissacarídeos/biossíntese , Polissacarídeos/química , CinéticaRESUMO
Due to its detrimental impact on human health and the environment, regulations demand ultralow sulfur levels on fossil fuels, in particular in diesel. However, current desulfurization techniques are expensive and cannot efficiently remove heteroaromatic sulfur compounds, which are abundant in crude oil and concentrate in the diesel fraction after distillation. Biodesulfurization via the four enzymes of the metabolic 4S pathway of the bacterium Rhodococcus erythropolis (DszA-D) is a possible solution. However, the 4S pathway needs to operate at least 500 times faster for industrial applicability, a goal currently pursued through enzyme engineering. In this work, we unveil the catalytic mechanism of the flavin monooxygenase DszA. Surprisingly, we found that this enzyme follows a recently proposed atypical mechanism that passes through the formation of an N5OOH intermediate at the re side of the cofactor, aided by a well-defined, predominantly hydrophobic O2 pocket. Besides clarifying the unusual chemical mechanism of the complex DszA enzyme, with obvious implications for understanding the puzzling chemistry of flavin-mediated catalysis, the result is crucial for the rational engineering of DszA, contributing to making biodesulfurization attractive for the oil refining industry.
Assuntos
Biocatálise , Rhodococcus , Rhodococcus/enzimologia , Rhodococcus/metabolismo , Modelos Moleculares , Enxofre/metabolismo , Enxofre/química , Oxigenases de Função Mista/metabolismo , Oxigenases de Função Mista/química , Carbono/química , Carbono/metabolismoRESUMO
BACKGROUND: Nitrile Hydratase (NHase) is one of the most important industrial enzyme widely used in the petroleum exploitation field. The enzyme, composed of two unrelated α- and ß-subunits, catalyzes the conversion of acrylonitrile to acrylamide, releasing a significant amount of heat and generating the organic solvent product, acrylamide. Both the heat and acrylamide solvent have an impact on the structural stability of NHase and its catalytic activity. Therefore, enhancing the stress resistance of NHase to toxic substances is meaningful for the petroleum industry. METHODS AND RESULTS: To improve the thermo-stability and acrylamide tolerance of NHase, the two subunits were fused in vivo using SpyTag and SpyCatcher, which were attached to the termini of each subunit in various combinations. Analysis of the engineered strains showed that the C-terminus of ß-NHase is a better fusion site than the N-terminus, while the C-terminus of α-NHase is the most suitable site for fusion with a larger protein. Fusion of SpyTag and SpyCatcher to the C-terminus of ß-NHase and α-NHase, respectively, led to improved acrylamide tolerance and a slight enhancement in the thermo-stability of one of the engineered strains, NBSt. CONCLUSION: These results indicate that in vivo ligation of different subunits using SpyTag/SpyCatcher is a valuable strategy for enhancing subunit interaction and improving stress tolerance.
Assuntos
Hidroliases , Rhodococcus , Rhodococcus/enzimologia , Rhodococcus/genética , Hidroliases/metabolismo , Hidroliases/genética , Hidroliases/química , Estabilidade Enzimática , Estresse Fisiológico , Acrilamida/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Subunidades Proteicas/metabolismo , Subunidades Proteicas/genéticaRESUMO
Cholesterol oxidases (ChOxes) are enzymes that catalyze the oxidation of cholesterol to cholest-4-en-3-one. These enzymes find wide applications across various diagnostic and industrial settings. In addition, as a pathogenic factor of several bacteria, they have significant clinical implications. The current classification system for ChOxes is based on the type of bond connecting FAD to the apoenzyme, which does not adequately illustrate the enzymatic and structural characteristics of these proteins. In this study, we have adopted an integrative approach, combining evolutionary analysis, classic enzymatic techniques and computational approaches, to elucidate the distinct features of four various ChOxes from Rhodococcus sp. (RCO), Cromobacterium sp. (CCO), Pseudomonas aeruginosa (PCO) and Burkhoderia cepacia (BCO). Comparative and evolutionary analysis of substrate-binding domain (SBD) and FAD-binding domain (FBD) helped to reveal the origin of ChOxes. We discovered that all forms of ChOxes had a common ancestor and that the structural differences evolved later during divergence. Further examination of amino acid variations revealed SBD as a more variable compared to FBD independently of FAD coupling mechanism. Revealed differences in amino acid positions turned out to be critical in determining common for ChOxes properties and those that account for the individual differences in substrate specificity. A novel look with the help of chemical descriptors on found distinct features were sufficient to attempt an alternative classification system aimed at application approach. While univocal characteristics necessary to establish such a system remain elusive, we were able to demonstrate the substrate and protein features that explain the differences in substrate profile.
Assuntos
Proteínas de Bactérias , Colesterol Oxidase , Especificidade por Substrato , Colesterol Oxidase/química , Colesterol Oxidase/metabolismo , Colesterol Oxidase/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Rhodococcus/enzimologia , Pseudomonas aeruginosa/enzimologia , Evolução Molecular , Sequência de Aminoácidos , Domínios Proteicos , Flavina-Adenina Dinucleotídeo/metabolismo , Flavina-Adenina Dinucleotídeo/química , FilogeniaRESUMO
Propanil residues can contaminate habitats where microbial degradation is predominant. In this study, an efficient propanil-degrading strain C-1 was isolated from paddy and identified as Rhodococcus sp. It can completely degrade 10 µg/L-150 mg/L propanil within 0.33-10 h via the hydrolysis of the amide bond, forming 3,4-dichloroaniline. A novel bifunctional amidase, PamC, was identified in strain C-1. PamC can catalyze the hydrolysis of the amide bond of propanil to produce 3,4-dichloroaniline as well as the hydrolysis of the ester bonds of aryloxyphenoxypropionate herbicides (APPHs, clodinafop-propargyl, cyhalofop-butyl, fenoxaprop-p-ethyl, fluazifop-p-butyl, haloxyfop-p-methyl, and quizalofop-p-ethyl) to form aryloxyphenoxypropionic acids. Molecular docking and site-directed mutagenesis confirmed that the catalytic triad Lys82-Ser157-Ser181 was the active center for PamC to hydrolyze propanil and cyhalofop-butyl. This study presents a novel bifunctional amidase with capabilities for both amide and ester bond hydrolysis and enhances our understanding of the molecular mechanisms underlying the degradation of propanil and APPHs.
Assuntos
Amidoidrolases , Proteínas de Bactérias , Biodegradação Ambiental , Herbicidas , Propanil , Rhodococcus , Rhodococcus/enzimologia , Rhodococcus/genética , Rhodococcus/metabolismo , Herbicidas/metabolismo , Herbicidas/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Propanil/metabolismo , Propanil/química , Amidoidrolases/metabolismo , Amidoidrolases/química , Amidoidrolases/genética , Simulação de Acoplamento Molecular , Hidrólise , BiocatáliseRESUMO
Phthalate esters (PAEs) are widely used as plasticizers and cause serious complex pollution problem in environment. Thus, strains with efficient ability to simultaneously degrade various PAEs are required. In this study, a newly isolated strain Rhodococcus sp. AH-ZY2 can degrade 500 mg/L Di-n-octyl phthalate completely within 16 h and other 500 mg/L PAEs almost completely within 48 h at 37 °C, 180 rpm, and 2 % (v/v) inoculum size of cultures with a OD600 of 0.8. OD600 = 0.8, 2 % (v/v). Twenty genes in its genome were annotated as potential esterase and four of them (3963, 4547, 5294 and 5359) were heterogeneously expressed and characterized. Esterase 3963 and 4547 is a type I PAEs esterase that hydrolyzes PAEs to phthalate monoesters. Esterase 5294 is a type II PAEs esterase that hydrolyzes phthalate monoesters to phthalate acid (PA). Esterase 5359 is a type III PAEs esterase that simultaneously degrades various PAEs to PA. Molecular docking results of 5359 suggested that the size and indiscriminate binding feature of spacious substrate binding pocket may contribute to its substrate versatility. AH-ZY2 is a potential strain for efficient remediation of PAEs complex pollution in environment. It is first to report an esterase that can efficiently degrade mixed various PAEs.
Assuntos
Biodegradação Ambiental , Esterases , Ésteres , Simulação de Acoplamento Molecular , Ácidos Ftálicos , Rhodococcus , Rhodococcus/metabolismo , Rhodococcus/genética , Rhodococcus/enzimologia , Ácidos Ftálicos/metabolismo , Ácidos Ftálicos/química , Esterases/metabolismo , Esterases/genética , Ésteres/metabolismo , Ésteres/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Plastificantes/metabolismoRESUMO
Uracil-thymine dehydrogenase (UTDH), which catalyzes the irreversible oxidation of uracil to barbituric acid in oxidative pyrimidine metabolism, was purified from Rhodococcus erythropolis JCM 3132. The finding of unusual stabilizing conditions (pH 11, in the presence of NADP+ or NADPH) enabled the enzyme purification. The purified enzyme was a heteromer consisting of three different subunits. The enzyme catalyzed oxidation of uracil to barbituric acid with artificial electron acceptors such as methylene blue, phenazine methosulfate, benzoquinone, and α-naphthoquinone; however, NAD+, NADP+, flavin adenine dinucleotide, and flavin mononucleotide did not serve as electron acceptors. The enzyme acted not only on uracil and thymine but also on 5-halogen-substituted uracil and hydroxypyrimidine (pyrimidone), while dihydropyrimidine, which is an intermediate in reductive pyrimidine metabolism, and purine did not serve as substrates. The activity of UTDH was enhanced by cerium ions, and this activation was observed with all combinations of substrates and electron acceptors.
Assuntos
Oxirredução , Pirimidinas , Rhodococcus , Uracila , Uracila/metabolismo , Uracila/química , Pirimidinas/metabolismo , Rhodococcus/enzimologia , NADP/metabolismo , Azul de Metileno/metabolismo , Azul de Metileno/química , Barbitúricos/metabolismo , Barbitúricos/química , Benzoquinonas/metabolismo , Benzoquinonas/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Concentração de Íons de Hidrogênio , Timina/metabolismo , Timina/química , Especificidade por Substrato , Metilfenazônio Metossulfato/metabolismo , Metilfenazônio Metossulfato/químicaRESUMO
Isoamyl fatty acid esters (IAFEs) are widely used as fruity flavor compounds in the food industry. In this study, various IAFEs were synthesized from isoamyl alcohol and various fatty acids using a cutinase enzyme (Rcut) derived from Rhodococcus bacteria. Rcut was immobilized on methacrylate divinylbenzene beads and used to synthesize isoamyl acetate, butyrate, hexanoate, octanoate, and decanoate. Among them, Rcut synthesized isoamyl butyrate (IAB) most efficiently. Docking model studies showed that butyric acid was the most suitable substrate in terms of binding energy and distance from the active site serine (Ser114) γ-oxygen. Up to 250 mM of IAB was synthesized by adjusting reaction conditions such as substrate concentration, reaction temperature, and reaction time. When the enzyme reaction was performed by reusing the immobilized enzyme, the enzyme activity was maintained at least six times. These results demonstrate that the immobilized Rcut enzyme can be used in the food industry to synthesize a variety of fruity flavor compounds, including IAB.
Assuntos
Hidrolases de Éster Carboxílico , Enzimas Imobilizadas , Aromatizantes , Simulação de Acoplamento Molecular , Rhodococcus , Enzimas Imobilizadas/metabolismo , Enzimas Imobilizadas/química , Rhodococcus/enzimologia , Rhodococcus/metabolismo , Aromatizantes/metabolismo , Aromatizantes/química , Hidrolases de Éster Carboxílico/metabolismo , Hidrolases de Éster Carboxílico/química , Ésteres/metabolismo , Ésteres/química , Pentanóis/metabolismo , Pentanóis/química , Ácidos Graxos/metabolismo , Ácidos Graxos/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Temperatura , Especificidade por Substrato , Ácido Butírico/metabolismo , Ácido Butírico/química , Domínio CatalíticoRESUMO
The strain JPL-2, capable of degrading fenoxaprop-P-ethyl (FE), was isolated from the soil of a wheat field and identified as Rhodococcus ruber. This strain could utilize FE as its sole carbon source and degrade 94.6% of 100 mg L−1 FE in 54 h. Strain JPL-2 could also degrade other aryloxyphenoxy propanoate (AOPP) herbicides. The initial step of the degradation pathway is to hydrolyze the carboxylic acid ester bond. A novel esterase gene feh, encoding the FE-hydrolyzing carboxylesterase (FeH) responsible for this initial step, was cloned from strain JPL-2. Its molecular mass was approximately 39 kDa, and the catalytic efficiency of FeH followed the order of FE > quizalofop-P-ethyl > clodinafop-propargyl > cyhalofop-butyl > fluazifop-P-butyl > haloxyfop-P-methyl > diclofop-methy, which indicated that the chain length of the alcohol moiety strongly affected the hydrolysis activity of the FeH toward AOPP herbicides.