RESUMEN
The lipoxygenase cascade in plants is a source of oxylipins (oxidized fatty acid derivatives), which play an important role in regulatory processes and formation of plant response to stress factors. Some of the most common enzymes of the lipoxygenase cascade are 13-specific hydroperoxide lyases (HPLs, also called hemiacetal synthases) of the CYP74B subfamily. In this work, we identified and cloned the CYP74B34 gene from carrot (Daucus carota L.) and described the biochemical properties of the corresponding recombinant enzyme. The CYP74B34 enzyme was active towards 9- and 13-hydroperoxides of linoleic (9-HPOD and 13-HPOD, respectively) and α-linolenic (9-HPOT and 13-HPOT, respectively) acids. CYP74B34 specifically converted 9-HPOT and 13-HPOT into aldo acids (HPL products). The transformation of 13-HPOD led to the formation of aldo acids and epoxyalcohols [products of epoxyalcohol synthase (EAS) activity] as major and minor products, respectively. At the same time, conversion of 9-HPOD resulted in the formation of epoxyalcohols as the main products and aldo acids as the minor ones. Therefore, CYP74B34 is the first enzyme with a double HPL/EAS activity described in carrot. The presence of these catalytic activities was confirmed by analysis of the oxylipin profiles for the roots from young seedlings and mature plants. In addition, we substituted amino acid residues in one of the catalytically essential sites of the CYP74B34 and CYP74B33 proteins and investigated the properties of the obtained mutant enzymes.
Asunto(s)
Aldehído-Liasas , Sistema Enzimático del Citocromo P-450 , Daucus carota , Proteínas de Plantas , Daucus carota/enzimología , Daucus carota/genética , Daucus carota/metabolismo , Aldehído-Liasas/metabolismo , Aldehído-Liasas/genética , Aldehído-Liasas/química , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Peróxidos Lipídicos/metabolismo , Especificidad por Sustrato , Secuencia de Aminoácidos , Ácidos LinoleicosRESUMEN
Queuosine (Q) stands out as the sole tRNA modification that can be synthesized via salvage pathways. Comparative genomic analyses identified specific bacteria that showed a discrepancy between the projected Q salvage route and the predicted substrate specificities of the two identified salvage proteins: (1) the distinctive enzyme tRNA guanine-34 transglycosylase (bacterial TGT, or bTGT), responsible for inserting precursor bases into target tRNAs; and (2) queuosine precursor transporter (QPTR), a transporter protein that imports Q precursors. Organisms such as the facultative intracellular pathogen Bartonella henselae, which possess only bTGT and QPTR but lack predicted enzymes for converting preQ1 to Q, would be expected to salvage the queuine (q) base, mirroring the scenario for the obligate intracellular pathogen Chlamydia trachomatis. However, sequence analyses indicate that the substrate-specificity residues of their bTGTs resemble those of enzymes inserting preQ1 rather than q. Intriguingly, MS analyses of tRNA modification profiles in B. henselae reveal trace amounts of preQ1, previously not observed in a natural context. Complementation analysis demonstrates that B. henselae bTGT and QPTR not only utilize preQ1, akin to their Escherichia coli counterparts, but can also process q when provided at elevated concentrations. The experimental and phylogenomic analyses suggest that the Q pathway in B. henselae could represent an evolutionary transition among intracellular pathogens - from ancestors that synthesized Q de novo to a state prioritizing the salvage of q. Another possibility that will require further investigations is that the insertion of preQ1 confers fitness advantages when B. henselae is growing outside a mammalian host.
Asunto(s)
Bartonella henselae , Nucleósido Q , Nucleósido Q/metabolismo , Nucleósido Q/genética , Bartonella henselae/genética , Bartonella henselae/metabolismo , Bartonella henselae/enzimología , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Evolución Molecular , Especificidad por Sustrato , Guanina/análogos & derivadosRESUMEN
Lacto-N-neotetraose (LNnT), as a neutral core structure within human milk oligosaccharides (HMOs), has garnered widespread attention due to its exceptional physiological functions. In the process of LNnT synthesis using cellular factory approaches, substrate promiscuity of glycosyltransferases leads to the production of longer oligosaccharide derivatives. Here, rational modification of ß1,3-N-acetylglucosaminyltransferase from Neisseria meningitidis (LgtA) effectively decreased the concentration of long-chain LNnT derivatives. Specifically, the optimal ß1,4-galactosyltransferase (ß1,4-GalT) was selected from seven known candidates, enabling the efficient synthesis of LNnT in Escherichia coli BL21(DE3). Furthermore, the influence of lactose concentration on the distribution patterns of LNnT and its longer derivatives was investigated. The modification of LgtA was conducted with computational assistance, involving alanine scanning based on molecular docking to identify the substrate binding pocket and implementing large steric hindrance on crucial amino acids to obstruct LNnT entry. The implementation of saturation mutagenesis at positions 223 and 228 of LgtA yielded advantageous mutant variants that did not affect LNnT synthesis while significantly reducing the production of longer oligosaccharide derivatives. The most effective mutant, N223I, reduced the molar ratio of long derivatives by nearly 70 %, showcasing promising prospects for LNnT production with diminished byproducts.
Asunto(s)
N-Acetilglucosaminiltransferasas , Neisseria meningitidis , Oligosacáridos , Neisseria meningitidis/enzimología , N-Acetilglucosaminiltransferasas/metabolismo , N-Acetilglucosaminiltransferasas/genética , Oligosacáridos/química , Oligosacáridos/síntesis química , Simulación del Acoplamiento Molecular , Escherichia coli/genética , Especificidad por Sustrato , Lactosa/análogos & derivados , Lactosa/metabolismo , Lactosa/química , HumanosRESUMEN
Streptococcus suis (S. suis) is an important swine bacterial pathogen and causes human infections, leading to a wide range of diseases. However, the role of 5'-nucleotidases in its virulence remains to be fully elucidated. Herein, we identified four cell wall-anchored 5'-nucleotidases (Snts) within S. suis, named SntA, SntB, SntC, and SntD, each displaying similar domains yet exhibiting low sequence homology. The malachite green reagent and HPLC assays demonstrated that these recombinant enzymes are capable of hydrolysing ATP, ADP, and AMP into adenosine (Ado), with the hierarchy of catalytic efficiency being SntC>SntB>SntA>SntD. Moreover, comprehensive enzymatic activity assays illustrated slight variances in substrate specificity, pH tolerance, and metal ion requirements, yet highlighted a conserved substrate-binding pocket, His-Asp catalytic dyad, metal, and phosphate-binding sites across Snts, with the exception of SntA. Through bactericidal assays and murine infection assays involving in site-mutagenesis strains, it was demonstrated that SntB and SntC collaboratively enhance bacterial survivability within whole blood and polymorphonuclear leukocytes (PMNs) via the Ado-A2aR pathway in vitro, and within murine blood and organs in vivo. This suggests a direct correlation between enzymatic activity and enhancement of bacterial survival and virulence. Collectively, S. suis 5'-nucleotidases additively contribute to the generation of adenosine, influencing susceptibility within blood and PMNs, and enhancing survival within blood and organs in vivo. This elucidation of their integral functions in the pathogenic process of S. suis not only enhances our comprehension of bacterial virulence mechanisms, but also illuminates new avenues for therapeutic intervention aimed at curbing S. suis infections.
Asunto(s)
5'-Nucleotidasa , Adenosina , Modelos Animales de Enfermedad , Evasión Inmune , Infecciones Estreptocócicas , Streptococcus suis , Animales , Streptococcus suis/patogenicidad , Streptococcus suis/enzimología , Streptococcus suis/inmunología , Streptococcus suis/genética , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/inmunología , 5'-Nucleotidasa/metabolismo , Ratones , Adenosina/metabolismo , Virulencia , Infecciones Estreptocócicas/microbiología , Infecciones Estreptocócicas/inmunología , Femenino , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/inmunología , Neutrófilos/inmunología , Neutrófilos/microbiología , Ratones Endogámicos BALB C , Especificidad por SustratoRESUMEN
Thiol dioxygenases (TDOs) are nonheme Fe(II)dependent enzymes that catalyze the O2-dependent oxidation of thiol substrates to their corresponding sulfinic acids. Six classes of TDOs have thus far been identified and two, cysteine dioxygenase (CDO) and cysteamine dioxygenase (ADO), are found in eukaryotes. All TDOs belong to the cupin superfamily of enzymes, which share a common ßbarrel fold and two cupin motifs: G(X)5HXH(X)3-6E(X)6G and G(X)5-7PXG(X)2H(X)3N. Crystal structures of TDOs revealed that these enzymes contain a relatively rare, neutral 3His ironbinding facial triad. Despite this shared metal-binding site, TDOs vary greatly in their secondary coordination spheres. Sitedirected mutagenesis has been used extensively to explore the impact of changes in secondary sphere residues on substrate specificity and enzymatic efficiency. This chapter summarizes site-directed mutagenesis studies of eukaryotic TDOs, focusing on the tools and practicality of nonstandard amino acid incorporation.
Asunto(s)
Aminoácidos , Dioxigenasas , Mutagénesis Sitio-Dirigida , Dioxigenasas/química , Dioxigenasas/metabolismo , Dioxigenasas/genética , Aminoácidos/metabolismo , Aminoácidos/química , Especificidad por Sustrato , Cisteína-Dioxigenasa/química , Cisteína-Dioxigenasa/metabolismo , Cisteína-Dioxigenasa/genética , Compuestos de Sulfhidrilo/metabolismo , Compuestos de Sulfhidrilo/química , Humanos , Animales , Modelos MolecularesRESUMEN
The inversion of substrate size specificity is an evolutionary roadblock for proteins. The Duf4243 dioxygenases GedK and BTG13 are known to catalyze the aromatic cleavage of bulky tricyclic hydroquinone. In this study, we discover a Duf4243 dioxygenase PaD that favors small monocyclic hydroquinones from the penicillic-acid biosynthetic pathway. Sequence alignments between PaD and GedK and BTG13 suggest PaD has three additional motifs, namely motifs 1-3, distributed at different positions in the protein sequence. X-ray crystal structures of PaD with the substrate at high resolution show motifs 1-3 determine three loops (loops 1-3). Most intriguing, loops 1-3 stack together at the top of the pocket, creating a lid-like tertiary structure with a narrow channel and a clearly constricted opening. This drastically changes the substrate specificity by determining the entry and binding of much smaller substrates. Further genome mining suggests Duf4243 dioxygenases with motifs 1-3 belong to an evolutionary branch that is extensively involved in the biosynthesis of natural products and has the ability to degrade diverse monocyclic hydroquinone pollutants. This study showcases how natural enzymes alter the substrate specificity fundamentally by incorporating new small motifs, with a fixed overall scaffold-architecture. It will also offer a theoretical foundation for the engineering of substrate specificity in enzymes and act as a guide for the identification of aromatic dioxygenases with distinct substrate specificities.
Asunto(s)
Secuencias de Aminoácidos , Dioxigenasas , Especificidad por Sustrato , Dioxigenasas/metabolismo , Dioxigenasas/genética , Dioxigenasas/química , Cristalografía por Rayos X , Hidroquinonas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Secuencia de Aminoácidos , Modelos Moleculares , Alineación de SecuenciaRESUMEN
The inhibitor of κB (IκB) kinase (IKK) is a central regulator of NF-κB signaling. All IKK complexes contain hetero- or homodimers of the catalytic IKKß and/or IKKα subunits. Here, we identify a YDDΦxΦ motif, which is conserved in substrates of canonical (IκBα, IκBß) and alternative (p100) NF-κB pathways, and which mediates docking to catalytic IKK dimers. We demonstrate a quantitative correlation between docking affinity and IKK activity related to IκBα phosphorylation/degradation. Furthermore, we show that phosphorylation of the motif's conserved tyrosine, an event previously reported to promote IκBα accumulation and inhibition of NF-κB gene expression, suppresses the docking interaction. Results from integrated structural analyzes indicate that the motif binds to a groove at the IKK dimer interface. Consistently, suppression of IKK dimerization also abolishes IκBα substrate binding. Finally, we show that an optimized bivalent motif peptide inhibits NF-κB signaling. This work unveils a function for IKKα/ß dimerization in substrate motif recognition.
Asunto(s)
Secuencias de Aminoácidos , Quinasa I-kappa B , FN-kappa B , Multimerización de Proteína , Quinasa I-kappa B/metabolismo , Quinasa I-kappa B/química , Quinasa I-kappa B/genética , Humanos , FN-kappa B/metabolismo , Fosforilación , Unión Proteica , Transducción de Señal , Inhibidor NF-kappaB alfa/metabolismo , Inhibidor NF-kappaB alfa/genética , Simulación del Acoplamiento Molecular , Células HEK293 , Especificidad por SustratoRESUMEN
Jumonji-C (JmjC) domain-containing protein 7 (JMJD7) is a human Fe(II) and 2-oxoglutarate dependent oxygenase that catalyzes stereospecific C3-hydroxylation of lysyl-residues in developmentally regulated GTP binding proteins 1 and 2 (DRG1/2). We report studies exploring a diverse set of lysine derivatives incorporated into the DRG1 peptides as potential human JMJD7 substrates and inhibitors. The results indicate that human JMJD7 has a relatively narrow substrate scope beyond lysine compared to some other JmjC hydroxylases and lysine-modifying enzymes. The geometrically constrained (E)-dehydrolysine is an efficient alternative to lysine for JMJD7-catalyzed C3-hydroxylation. γ-Thialysine and γ-azalysine undergo C3-hydroxylation, followed by degradation to formylglycine. JMJD7 also catalyzes the S-oxidation of DRG1-derived peptides possessing methionine and homomethionine residues in place of lysine. Inhibition assays show that DRG1 variants possessing cysteine/selenocysteine instead of the lysine residue efficiently inhibit JMJD7 via cross-linking. The overall results inform on the substrate selectivity and inhibition of human JMJD7, which will help enable the rational design of selective small-molecule and peptidomimetic inhibitors of JMJD7.
Asunto(s)
Histona Demetilasas con Dominio de Jumonji , Humanos , Histona Demetilasas con Dominio de Jumonji/química , Histona Demetilasas con Dominio de Jumonji/metabolismo , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/genética , Especificidad por Sustrato , Lisina/química , Lisina/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , HidroxilaciónRESUMEN
PE/PPE proteins secreted by the ESX-5 type VII secretion system constitute a major protein repertoire in pathogenic mycobacteria and are essential for bacterial survival, pathogenicity, and host-pathogen interaction; however, little is known about their expression and secretion. The scarcity of arginine and lysine residues in PE/PPE protein sequences and the high homology of their N-terminal domains limit protein identification using classical trypsin-based proteomic methods. This study used endoproteinase AspN and trypsin to characterize the proteome of Mycobacterium marinum. Twenty-seven PE/PPE proteins were uniquely identified in AspN digests, especially PE_PGRS proteins. These treatments allowed the identification of approximately 50% of the PE/PPE pool encoded in the genome. Moreover, EspG5 pulldown assays retrieved 44 ESX-5-associated PPE proteins, covering 85% of the PPE pool in the identified proteome. The identification of PE/PE_PGRS proteins in the EspG5 interactome suggested the presence of PE-PPE pairs. The correlation analysis between protein abundance and phylogenetic relationships found potential PE/PPE pairs, indicating the presence of multiple PE/PE_PGRS partners in one PPE. We validated that EspG5 interacted with PPE31 and PPE32 and mapped critical residues for complex formation. The modified proteomic platform increases the coverage of PE/PPE proteins and elucidates the expression and localization of these proteins.
Asunto(s)
Proteínas Bacterianas , Mycobacterium marinum , Proteoma , Mycobacterium marinum/metabolismo , Mycobacterium marinum/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteoma/metabolismo , Proteómica/métodos , Filogenia , Sistemas de Secreción Tipo VII/metabolismo , Sistemas de Secreción Tipo VII/genética , Especificidad por SustratoRESUMEN
Phenylpropanoid sucrose esters are a large and important group of natural substances with significant therapeutic potential. This work describes a pilot study of the enzymatic hydroxycinnamoylation of sucrose and its derivatives which was carried out with the aim of obtaining precursors of natural phenylpropanoid sucrose esters, e.g., vanicoside B. In addition to sucrose, some chemically prepared sucrose acetonides and substituted 3'-O-cinnamates were subjected to enzymatic transesterification with vinyl esters of coumaric, ferulic and 3,4,5-trimethoxycinnamic acid. Commercial enzyme preparations of Lipozyme TL IM lipase and Pentopan 500 BG exhibiting feruloyl esterase activity were tested as biocatalysts in these reactions. The substrate specificity of the used biocatalysts for the donor and acceptor as well as the regioselectivity of the reactions were evaluated and discussed. Surprisingly, Lipozyme TL IM catalyzed the cinnamoylation of sucrose derivatives more to the 1'-OH and 4'-OH positions than to the 6'-OH when the 3'-OH was free and the 6-OH was blocked by isopropylidene. In this case, Pentopan reacted comparably to 1'-OH and 6'-OH positions. If sucrose 3'-O-coumarate was used as an acceptor, in the case of feruloylation with Lipozyme in CH3CN, 6-O-ferulate was the main product (63%). Pentopan feruloylated sucrose 3'-O-coumarate comparably well at the 6-OH and 6'-OH positions (77%). When a proton-donor solvent was used, migration of the 3'-O-cinnamoyl group from fructose to the 2-OH position of glucose was observed. The enzyme hydroxycinnamoylations studied can shorten the targeted syntheses of various phenylpropanoid sucrose esters.
Asunto(s)
Ácidos Cumáricos , Sacarosa , Sacarosa/química , Sacarosa/metabolismo , Ácidos Cumáricos/química , Ácidos Cumáricos/metabolismo , Lipasa/metabolismo , Lipasa/química , Cinamatos/química , Cinamatos/metabolismo , Especificidad por Sustrato , Esterificación , Hidrolasas de Éster Carboxílico/metabolismo , Hidrolasas de Éster Carboxílico/química , Ésteres/química , Ésteres/metabolismo , BiocatálisisRESUMEN
Sucrose phosphorylase (SPase), a member of the glycoside hydrolase GH13 family, possesses the ability to catalyze the hydrolysis of sucrose to generate α-glucose-1-phosphate and can also glycosylate diverse substrates, showcasing a wide substrate specificity. This enzyme has found extensive utility in the fields of food, medicine, and cosmetics, and has garnered significant attention as a focal point of research in transglycosylation enzymes. Nevertheless, SPase encounters numerous obstacles in industrial settings, including low enzyme yield, inadequate thermal stability, mixed regioselectivity, and limited transglycosylation activity. In-depth exploration of efficient expression strategies and molecular modifications based on the crystal structure and functional information of SPase is now a critical research priority. This paper systematically reviews the source microorganisms, crystal structure, and catalytic mechanism of SPase, summarizes diverse heterologous expression systems based on expression hosts and vectors, and examines the application and molecular modification progress of SPase in synthesizing typical glycosylated products. Additionally, it anticipates the broad application prospects of SPase in industrial production and related research fields, laying the groundwork for its engineering modification and industrial application.
Asunto(s)
Glucosiltransferasas , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Glucosiltransferasas/química , Glucosiltransferasas/biosíntesis , Glicosilación , Especificidad por Sustrato , Expresión GénicaRESUMEN
Determination of substrate binding affinity (Kd) is critical to understanding enzyme function. An extensive number of methods have been developed and employed to study ligand/substrate binding, but the best approach depends greatly on the substrate and the enzyme in question. Below we describe how to measure the Kd of BesD, a non-heme iron halogenase, for its native substrate lysine using equilibrium dialysis coupled with High Performance Liquid Chromatography (HPLC) for subsequent detection. This method can be performed in anaerobic glove bag settings. It requires readily available HPLC instrumentation for ligand quantitation and is adaptable to meet the needs of a variety of substrate affinity measurements.
Asunto(s)
Diálisis , Cromatografía Líquida de Alta Presión/métodos , Especificidad por Sustrato , Diálisis/métodos , Unión Proteica , Pruebas de Enzimas/métodos , Pruebas de Enzimas/instrumentación , Cinética , Lisina/metabolismo , Lisina/química , Oxidorreductasas/metabolismo , Oxidorreductasas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Hierro/metabolismo , Hierro/químicaRESUMEN
The acquisition of new RNA functions through evolutionary processes was essential for the diversification of RNA-based primordial biology and its subsequent transition to modern biology. However, the mechanisms by which RNAs access new functions remain unclear. Do RNA enzymes need completely new folds to support new but related functions, or is reoptimization of the active site sufficient? What are the roles of neutral and adaptive mutations in evolutionary innovation? Here, we address these questions experimentally by focusing on the evolution of substrate specificity in RNA-catalyzed RNA assembly. We use directed in vitro evolution to show that a ligase ribozyme that uses prebiotically relevant 5'-phosphorimidazole-activated substrates can be evolved to catalyze ligation with substrates that are 5'-activated with the biologically relevant triphosphate group. Interestingly, despite catalyzing a related reaction, the new ribozyme folds into a completely new structure and exhibits promiscuity by catalyzing RNA ligation with both triphosphate and phosphorimidazole-activated substrates. Although distinct in sequence and structure, the parent phosphorimidazolide ligase and the evolved triphosphate ligase ribozymes can be connected by a series of point mutations where the intermediate sequences retain at least some ligase activity. The existence of a quasi-neutral pathway between these distinct ligase ribozymes suggests that neutral drift is sufficient to enable the acquisition of new substrate specificity, thereby providing opportunities for subsequent adaptive optimization. The transition from RNA-catalyzed RNA assembly using phosphorimidazole-activated substrates to triphosphate-activated substrates may have foreshadowed the later evolution of the protein enzymes that use monomeric triphosphates (nucleoside triphosphates, NTPs) for RNA synthesis.
Asunto(s)
Imidazoles , ARN Ligasa (ATP) , ARN Catalítico , ARN Catalítico/metabolismo , ARN Catalítico/química , ARN Catalítico/genética , Especificidad por Sustrato , Imidazoles/metabolismo , Imidazoles/química , ARN Ligasa (ATP)/metabolismo , ARN Ligasa (ATP)/química , ARN Ligasa (ATP)/genética , Evolución Molecular , Conformación de Ácido Nucleico , Dominio CatalíticoRESUMEN
Haloalkane dehalogenase, LinB, is a member of the α/ß hydrolase family of enzymes. It has a wide range of halogenated substrates, but, has been mostly studied in context of degradation of hexachlorocyclohexane (HCH) isomers, especially ß-HCH (5-12% of total HCH isomers), which is the most recalcitrant and persistent among all the HCH isomers. LinB was identified to directly act on ß-HCH in a one or two step transformation which decreases its toxicity manifold. Thereafter, many studies focused on LinB including its structure determination using X-ray crystallographic studies, structure comparison with other haloalkane dehalogenases, substrate specificity and kinetic studies, protein engineering and site-directed mutagenesis studies in search of better catalytic activity of the enzyme. LinB was mainly identified and characterized in bacteria belonging to sphingomonads. Detailed sequence comparison of LinB from different sphingomonads further revealed the residues critical for its activity and ability to catalyze either one or two step transformation of ß-HCH. Association of LinB with IS6100 elements is also being discussed in detail in sphingomonads. In this review, we summarized vigorous efforts done by different research groups on LinB for developing better bioremediation strategies against HCH contamination. Also, kinetic studies, protein engineering and site directed mutagenesis studies discussed here forms the basis of further exploration of LinB's role as an efficient enzyme in bioremediation projects.
Asunto(s)
Hexaclorociclohexano , Hidrolasas , Hidrolasas/metabolismo , Hidrolasas/genética , Hidrolasas/química , Hexaclorociclohexano/metabolismo , Especificidad por Sustrato , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Cinética , Biodegradación Ambiental , Cristalografía por Rayos X , Mutagénesis Sitio-Dirigida , Sphingomonas/enzimología , Sphingomonas/genética , Sphingomonas/metabolismoRESUMEN
Two multimodular endoglucanases in glycoside hydrolase family 5, ReCel5 and ElCel5, share 73% identity and exhibit similar modular structures: family 1 carbohydrate-binding module (CBM1); catalytic domain; CBMX2; module of unknown function. However, they differed in their biochemical properties and catalytic performance. ReCel5 showed optimal activity at pH 4.0 and 70 °C, maintaining stability at 70 °C (>80% activity). Conversely, ElCel5 is optimal at pH 3.0 and 50 °C (>50% activity at 50 °C). ElCel5 excels in degrading CMC-Na (256 U/mg vs 53 U/mg of ReCel5). Five domain-truncated (TM1-TM5) and four domain-replaced (RM1-RM4) mutants of ReCel5 with the counterparts of ElCel5 were constructed, and their enzymatic properties were compared with those of the wild type. Only RM1, with ElCel5-CBM1, displayed enhanced thermostability and activity. The hydrolysis of pretreated corn stover was reduced in most TM and RM mutants. Molecular dynamics simulations revealed interdomain interactions within the multimodular endoglucanase, potentially affecting its structural stability and complex biological catalytic processes.
Asunto(s)
Celulasa , Hidrólisis , Celulasa/química , Celulasa/metabolismo , Celulasa/genética , Celulosa/metabolismo , Celulosa/química , Dominios Proteicos , Dominio Catalítico , Especificidad por Sustrato , Zea mays/química , Simulación de Dinámica Molecular , Estabilidad de EnzimasRESUMEN
Prenylation of amino acids is a critical step for synthesizing building blocks of prenylated alkaloid family natural products, where the corresponding prenyltransferase that catalyzes prenylation on free l-histidine (l-His) has not yet been identified. Here, we first discovered and characterized a prenyltransferase FunA from the antifungal agent fungerin pathway that efficiently performs C4-dimethylallylation on l-His. Crystal structure-guided engineering of the prenyl-binding pocket of FunA, a single M181A mutation, successfully converted it into a C4-geranyltransferase. Furthermore, FunA and its variant FunA-M181A show broad substrate promiscuity toward substrates that vary in substituents of the imidazole ring. Our work furthers our knowledge of free amino acid prenyltransferase and expands the arsenal of alkylation biocatalysts for imidazole-containing small molecules.
Asunto(s)
Dimetilaliltranstransferasa , Histidina , Histidina/química , Histidina/metabolismo , Dimetilaliltranstransferasa/metabolismo , Dimetilaliltranstransferasa/química , Dimetilaliltranstransferasa/genética , Ingeniería de Proteínas , Modelos Moleculares , Especificidad por Sustrato , Imidazoles/química , Imidazoles/metabolismoRESUMEN
Mitomycins make up a class of natural molecules produced by Streptomyces with strong antibacterial and antitumor activities. MitM is a key postmitosane modification enzyme involved in mitomycin biosynthesis in Streptomyces caespitosus. This protein was previously suggested to catalyze the aziridinium methylation of mitomycin A and the mitomycin intermediate 9a-demethyl-mitomycin A as an N-methyltransferase. The structural basis for MitM to recognize cofactor S-adenosyl-l-methionine (SAM) and substrate mitomycin A is unknown. Here, we determined the crystal structures of apo-MitM and MitM-mitomycin A-S-adenosylhomocysteine (SAH) ternary complexes with resolutions of 2.23 and 2.80 Å, respectively. We found that MitM adopts a class I SAM-dependent methyltransferase fold and forms a homodimer in solution. Conformational changes in a series of residues involved in the formation of active pockets assist MitM in binding SAH and mitomycin A. In particular, the 28ALGAASLGE36 loop changes most significantly. When mitomycin A binds, the bending direction of this loop is reversed, changing the entrance of the active site from open to closed. This study provides structural insights into MitM's involvement in the postmitosane stage of mitomycin biosynthesis and provides a template for the engineering of methyltransferases.
Asunto(s)
Proteínas Bacterianas , Mitomicina , Streptomyces , Streptomyces/enzimología , Mitomicina/metabolismo , Mitomicina/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Especificidad por Sustrato , S-Adenosilmetionina/metabolismo , S-Adenosilmetionina/química , Metiltransferasas/metabolismo , Metiltransferasas/química , Modelos Moleculares , Dominio Catalítico , Conformación Proteica , S-Adenosilhomocisteína/metabolismo , S-Adenosilhomocisteína/químicaRESUMEN
Human terminal deoxynucleotidyl transferase (TdT) can catalyze template-independent DNA synthesis during the V(D)J recombination and DNA repair through nonhomologous end joining. The capacity for template-independent random addition of nucleotides to single-stranded DNA makes this polymerase useful in various molecular biological applications involving sequential stepwise synthesis of oligonucleotides using modified dNTP. Nonetheless, a serious limitation to the applications of this enzyme is strong selectivity of human TdT toward dNTPs in the order dGTP > dTTP ≈ dATP > dCTP. This study involved molecular dynamics to simulate a potential impact of amino acid substitutions on the enzyme's selectivity toward dNTPs. It was found that the formation of stable hydrogen bonds between a nitrogenous base and amino acid residues at positions 395 and 456 is crucial for the preferences for dNTPs. A set of single-substitution and double-substitution mutants at these positions was analyzed by molecular dynamics simulations. The data revealed two TdT mutants-containing either substitution D395N or substitutions D395N+E456N-that possess substantially equalized selectivity toward various dNTPs as compared to the wild-type enzyme. These results will enable rational design of TdT-like enzymes with equalized dNTP selectivity for biotechnological applications.
Asunto(s)
ADN Nucleotidilexotransferasa , Simulación de Dinámica Molecular , Humanos , ADN Nucleotidilexotransferasa/metabolismo , ADN Nucleotidilexotransferasa/química , ADN Nucleotidilexotransferasa/genética , Especificidad por Sustrato , Desoxirribonucleótidos/metabolismo , Desoxirribonucleótidos/química , Nucleótidos de Timina/metabolismo , Nucleótidos de Timina/química , Nucleótidos de Desoxicitosina/metabolismo , Nucleótidos de Desoxicitosina/química , Nucleótidos de Desoxiadenina/metabolismo , Nucleótidos de Desoxiadenina/química , Enlace de Hidrógeno , Nucleótidos de Desoxiguanina/metabolismo , Nucleótidos de Desoxiguanina/química , Sustitución de AminoácidosRESUMEN
Antibiotic resistance is one of most important health concerns nowadays, and ß-lactamases are the most important resistance determinants. These enzymes, based on their structural and functional characteristics, are grouped in four categories (A, B, C and D). We have solved the structure of PIB-1, a Pseudomonas aeruginosa chromosomally-encoded ß-lactamase, in its apo form and in complex with meropenem and zinc. These crystal structures show that it belongs to the Class C ß-lactamase group, although it shows notable differences, especially in the Ω- and P2-loops, which are important for the enzymatic activity. Functional analysis showed that PIB-1 is able to degrade carbapenems but not cephalosporins, the typical substrate of Class C ß-lactamases, and that its catalytic activity increases in the presence of metal ions, especially zinc. They do not bind to the active-site but they induce the formation of trimers that show an increased capacity for the degradation of the antibiotics, suggesting that this oligomer is more active than the other oligomeric species. While PIB-1 is structurally a Class C ß-lactamase, the low sequence conservation, substrate profile and its metal-dependence, prompts us to position this enzyme as the founder of a new group inside the Class C ß-lactamases. Consequently, its diversity might be wider than expected.
Asunto(s)
Carbapenémicos , Pseudomonas aeruginosa , Zinc , beta-Lactamasas , Pseudomonas aeruginosa/enzimología , beta-Lactamasas/química , beta-Lactamasas/metabolismo , Carbapenémicos/farmacología , Carbapenémicos/metabolismo , Carbapenémicos/química , Zinc/metabolismo , Zinc/química , Modelos Moleculares , Dominio Catalítico , Hidrólisis , Especificidad por Sustrato , Metales/metabolismo , Metales/química , Metales/farmacología , Relación Estructura-Actividad , Meropenem/farmacología , Meropenem/química , Meropenem/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos XRESUMEN
Resveratrol is a promising functional ingredient applied in food products. However, low bioavailability and poor water solubility, which can be improved by glycosylation, hinder its application. A uridine diphosphate-dependent glycosyltransferase (UGT) from Bacillus subtilis 168 (named UGTBS) presents potential application for resveratrol glycosylation; nonetheless, imprecise regioselectivity renders the synthesis of resveratrol-3-O-ß-D-glucoside (polydatin) difficult. Therefore, molecular evolution was applied to UGTBS. A triple mutant Y14I/I62G/M315W was developed for 3-OH glycosylation of resveratrol and polydatin accounted for 91% of the total product. Kinetic determination and molecular docking indicated that the enhancement of hydrogen bond interaction and altered conformation of the binding pocket increases the enzyme's affinity for the 3-OH group, stabilizing the enzyme-substrate intermediate and promoting polydatin formation. Furthermore, a fed-batch cascade reaction by periodic addition of resveratrol was conducted and nearly 20 mM polydatin was obtained. The mutant Y14I/I62G/M315W can be used for polydatin manufacture.