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1.
Biomolecules ; 11(9)2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34572512

RESUMO

Tuberculosis continues to be a major threat to the human population. Global efforts to eradicate the disease are ongoing but are hampered by the increasing occurrence of multidrug-resistant strains of Mycobacterium tuberculosis. Therefore, the development of new treatment, and the exploration of new druggable targets and treatment strategies, are of high importance. Rv0183/mtbMGL, is a monoacylglycerol lipase of M. tuberculosis and it is involved in providing fatty acids and glycerol as building blocks and as an energy source. Since the lipase is expressed during the dormant and active phase of an infection, Rv0183/mtbMGL is an interesting target for inhibition. In this work, we determined the crystal structures of a surface-entropy reduced variant K74A Rv0183/mtbMGL in its free form and in complex with a substrate mimicking inhibitor. The two structures reveal conformational changes in the cap region that forms a major part of the substrate/inhibitor binding region. We present a completely closed conformation in the free form and semi-closed conformation in the ligand-bound form. These conformations differ from the previously published, completely open conformation of Rv0183/mtbMGL. Thus, this work demonstrates the high conformational plasticity of the cap from open to closed conformations and provides useful insights into changes in the substrate-binding pocket, the target of potential small-molecule inhibitors.

2.
J Biol Chem ; 297(4): 101167, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34487759

RESUMO

ToxR represents an essential transcription factor of Vibrio cholerae, which is involved in the regulation of multiple, mainly virulence associated genes. Its versatile functionality as activator, repressor or coactivator suggests a complex regulatory mechanism, whose clarification is essential for a better understanding of the virulence expression system of V. cholerae. Here, we provide structural information elucidating the organization and binding behavior of the cytoplasmic DNA-binding domain of ToxR (cToxR), containing a winged helix-turn-helix (wHTH) motif. Our analysis reveals unexpected structural features of this domain expanding our knowledge of a poorly defined subfamily of wHTH proteins. cToxR forms an extraordinary long α-loop and furthermore has an additional C-terminal beta strand, contacting the N-terminus and thus leading to a compact fold. The identification of the exact interactions between ToxR and DNA contributes to a deeper understanding of this regulatory process. Our findings not only show general binding of the soluble cytoplasmic domain of ToxR to DNA, but also indicate a higher affinity for the toxT motif. These results support the current theory of ToxR being a "DNA-catcher" to enable binding of the transcription factor TcpP and thus activation of virulence-associated toxT transcription. Although, TcpP and ToxR interaction is assumed to be crucial in the activation of the toxT genes, we could not detect an interaction event of their isolated cytoplasmic domains. We therefore conclude that other factors are needed to establish this protein-protein interaction, e.g., membrane attachment, the presence of their full-length proteins and/or other intermediary proteins that may facilitate binding.


Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Fatores de Transcrição/química , Vibrio cholerae/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Sequências Hélice-Volta-Hélice , Domínios Proteicos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Genética , Vibrio cholerae/genética , Vibrio cholerae/metabolismo
3.
Science ; 373(6557): 871-876, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34282049

RESUMO

DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo-electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.


Assuntos
Aprendizado Profundo , Conformação Proteica , Dobramento de Proteína , Proteínas/química , Proteínas ADAM/química , Sequência de Aminoácidos , Simulação por Computador , Microscopia Crioeletrônica , Cristalografia por Raios X , Bases de Dados de Proteínas , Proteínas de Membrana/química , Modelos Moleculares , Complexos Multiproteicos/química , Redes Neurais de Computação , Subunidades Proteicas/química , Proteínas/fisiologia , Receptores Acoplados a Proteínas G/química , Esfingosina N-Aciltransferase/química
4.
Mol Microbiol ; 115(6): 1277-1291, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33368680

RESUMO

The transmembrane protein ToxR plays a key role in the virulence expression system of Vibrio cholerae. The activity of ToxR is dependent on its periplasmic sensor domain (ToxRp) and on the inner membrane protein ToxS. Herein, we present the Nuclear Magnetic Resonance NMR solution structure of the sensory ToxRp containing an intramolecular disulfide bond. The presented structural and dynamic experiments with reduced and oxidized ToxRp propose an explanation for the increased proteolytic sensitivity of reduced ToxR. Additionally, for the first time, we could identify the formation of a strong heterodimer complex between the periplasmic domains of ToxR and ToxS in solution. NMR interaction studies reveal that binding of ToxS is not dependent on the redox state of ToxR cysteines, and formed complexes are structurally similar. By monitoring the proteolytic cleavage of ToxRp with NMR, we additionally provide a direct evidence of ToxS protective function. Taken together our results suggest that ToxR activity is regulated by its stability which is, on the one hand, dependent on the redox states of its cysteines, influencing the stability of its fold, and on the other hand, on its interaction with ToxS, which binds independent on the cysteines and acts as a protection against proteases.

5.
ACS Catal ; 10(2): 1094-1101, 2020 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-32030315

RESUMO

The biocatalytic Friedel-Crafts acylation has been identified recently for the acetylation of resorcinol using activated acetic acid esters for the synthesis of acetophenone derivatives catalyzed by an acyltransferase. Because the wild-type enzyme is limited to acetic and propionic derivatives as the substrate, variants were designed to extend the substrate scope of this enzyme. By rational protein engineering, the key residue in the active site was identified which can be replaced to allow binding of bulkier acyl moieties. The single-point variant F148V enabled the transformation of previously inaccessible medium chain length alkyl and alkoxyalkyl carboxylic esters as donor substrates with up to 99% conversion and up to >99% isolated yield.

6.
Sci Rep ; 9(1): 2697, 2019 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-30804446

RESUMO

The enzyme 4-oxalocrotonate tautomerase shows remarkable catalytic versatility due to the secondary amine of its N-terminal proline moiety. In this work, we incorporated a range of proline analogues into the enzyme and examined the effects on structure and activity. While the structure of the enzyme remained unperturbed, its promiscuous Michael-type activity was severely affected. This finding demonstrates how atomic changes in a biocatalytic system can abolish its activity. Our work provides a toolbox for successful generation of enzyme variants with non-canonical catalytic proline analogues.


Assuntos
Isomerases/metabolismo , Animais , Biocatálise , Catálise , Humanos , Prolina/análogos & derivados , Prolina/metabolismo
7.
Chembiochem ; 20(1): 88-95, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30318713

RESUMO

C-C bond-forming reactions are key transformations for setting up the carbon frameworks of organic compounds. In this context, Friedel-Crafts acylation is commonly used for the synthesis of aryl ketones, which are common motifs in many fine chemicals and natural products. A bacterial multicomponent acyltransferase from Pseudomonas protegens (PpATase) catalyzes such Friedel-Crafts C-acylation of phenolic substrates in aqueous solution, reaching up to >99 % conversion without the need for CoA-activated reagents. We determined X-ray crystal structures of the native and ligand-bound complexes. This multimeric enzyme consists of three subunits: PhlA, PhlB, and PhlC, arranged in a Phl(A2 C2 )2 B4 composition. The structure of a reaction intermediate obtained from crystals soaked with the natural substrate 1-(2,4,6-trihydroxyphenyl)ethanone together with site-directed mutagenesis studies revealed that only residues from the PhlC subunits are involved in the acyl transfer reaction, with Cys88 very likely playing a significant role during catalysis. These structural and mechanistic insights form the basis of further enzyme engineering efforts directed towards enhancing the substrate scope of this enzyme.


Assuntos
Aciltransferases/química , Proteínas de Bactérias/química , Acilação , Aciltransferases/genética , Aciltransferases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Cristalografia por Raios X , Mutagênese Sítio-Dirigida , Mutação , Floroglucinol/análogos & derivados , Floroglucinol/química , Floroglucinol/metabolismo , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Pseudomonas/enzimologia
8.
J Biol Chem ; 293(44): 17021-17032, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30194285

RESUMO

Flavin-dependent enzymes catalyze many oxidations, including formation of ring structures in natural products. The gene cluster for biosynthesis of fumisoquins, secondary metabolites structurally related to isoquinolines, in the filamentous fungus Aspergillus fumigatus harbors a gene that encodes a flavoprotein of the amine oxidase family, termed fsqB (fumisoquin biosynthesis gene B). This enzyme catalyzes an oxidative ring closure reaction that leads to the formation of isoquinoline products. This reaction is reminiscent of the oxidative cyclization reported for berberine bridge enzyme and tetrahydrocannabinol synthase. Despite these similarities, amine oxidases and berberine bridge enzyme-like enzymes possess distinct structural properties, prompting us to investigate the structure-function relationships of FsqB. Here, we report the recombinant production and purification of FsqB, elucidation of its crystal structure, and kinetic analysis employing five putative substrates. The crystal structure at 2.6 Å resolution revealed that FsqB is a member of the amine oxidase family with a covalently bound FAD cofactor. N-methyl-dopa was the best substrate for FsqB and was completely converted to the cyclic isoquinoline product. The absence of the meta-hydroxyl group, as e.g. in l-N-methyl-tyrosine, resulted in a 25-fold lower rate of reduction and the formation of the demethylated product l-tyrosine, instead of a cyclic product. Surprisingly, FsqB did not accept the d-stereoisomer of N-methyltyrosine, in contrast to N-methyl-dopa, for which both stereoisomers were oxidized with similar rates. On the basis of the crystal structure and docking calculations, we postulate a substrate-dependent population of distinct binding modes that rationalizes stereospecific oxidation in the FsqB active site.


Assuntos
Aspergillus fumigatus/enzimologia , Di-Hidroxifenilalanina/metabolismo , Proteínas Fúngicas/química , Monoaminoxidase/química , Aspergillus fumigatus/genética , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Ciclização , Di-Hidroxifenilalanina/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cinética , Monoaminoxidase/genética , Monoaminoxidase/metabolismo , Família Multigênica , Oxirredução , Especificidade por Substrato
9.
Nucleic Acids Res ; 46(17): 9201-9219, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30060171

RESUMO

The dissemination of multi-resistant bacteria represents an enormous burden on modern healthcare. Plasmid-borne conjugative transfer is the most prevalent mechanism, requiring a type IV secretion system that enables bacteria to spread beneficial traits, such as resistance to last-line antibiotics, among different genera. Inc18 plasmids, like the Gram-positive broad host-range plasmid pIP501, are substantially involved in propagation of vancomycin resistance from Enterococci to methicillin-resistant strains of Staphylococcus aureus. Here, we identified the small cytosolic protein TraN as a repressor of the pIP501-encoded conjugative transfer system, since deletion of traN resulted in upregulation of transfer factors, leading to highly enhanced conjugative transfer. Furthermore, we report the complex structure of TraN with DNA and define the exact sequence of its binding motif. Targeting this protein-DNA interaction might represent a novel therapeutic approach against the spreading of antibiotic resistances.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Conjugação Genética , DNA Bacteriano/química , Enterococcus faecalis/genética , Proteínas de Escherichia coli/química , Plasmídeos/química , Sistemas de Secreção Tipo IV/genética , Sequência de Aminoácidos , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Sítios de Ligação , Cristalografia por Raios X , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Enterococcus faecalis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alinhamento de Sequência , Termodinâmica , Sistemas de Secreção Tipo IV/metabolismo , Vancomicina/farmacologia , Resistência a Vancomicina/genética
10.
Sci Rep ; 8(1): 8948, 2018 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-29895832

RESUMO

Monoacylglycerol lipases (MGLs) are enzymes that hydrolyze monoacylglycerol into a free fatty acid and glycerol. Fatty acids can be used for triacylglycerol synthesis, as energy source, as building blocks for energy storage, and as precursor for membrane phospholipids. In Mycobacterium tuberculosis, fatty acids also serve as precursor for polyketide lipids like mycolic acids, major components of the cellular envelope associated to resistance for drug. We present the crystal structure of the MGL Rv0183 from Mycobacterium tuberculosis (mtbMGL) in open conformation. The structure reveals remarkable similarities with MGL from humans (hMGL) in both, the cap region and the α/ß core. Nevertheless, mtbMGL could not be inhibited with JZL-184, a known inhibitor of hMGL. Docking studies provide an explanation why the activity of mtbMGL was not affected by the inhibitor. Our findings suggest that specific inhibition of mtbMGL from Mycobacterium tuberculosis, one of the oldest recognized pathogens, is possible without influencing hMGL.


Assuntos
Proteínas de Bactérias/metabolismo , Benzodioxóis/metabolismo , Monoacilglicerol Lipases/metabolismo , Mycobacterium tuberculosis/enzimologia , Piperidinas/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Benzodioxóis/química , Benzodioxóis/farmacologia , Cristalografia por Raios X , Cinética , Simulação de Dinâmica Molecular , Monoacilglicerol Lipases/antagonistas & inibidores , Monoacilglicerol Lipases/química , Monoglicerídeos/metabolismo , Piperidinas/química , Piperidinas/farmacologia , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato
11.
Front Microbiol ; 9: 250, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29515539

RESUMO

Carboxylate reductases (CARs, E.C. 1.2.1.30) generate aldehydes from their corresponding carboxylic acid with high selectivity. Little is known about the structure of CARs and their catalytically important amino acid residues. The identification of key residues for carboxylate reduction provides a starting point to gain deeper understanding of enzymatic carboxylate reduction. A multiple sequence alignment of CARs with confirmed activity recently identified in our lab and from the literature revealed a fingerprint of conserved amino acids. We studied the function of conserved residues by multiple sequence alignments and mutational replacements of these residues. In this study, single-site alanine variants of Neurospora crassa CAR were investigated to determine the contribution of conserved residues to the function, expressability or stability of the enzyme. The effect of amino acid replacements was investigated by analyzing enzymatic activity of the variants in vivo and in vitro. Supported by molecular modeling, we interpreted that five of these residues are essential for catalytic activity, or substrate and co-substrate binding. We identified amino acid residues having significant impact on CAR activity. Replacement of His 237, Glu 433, Ser 595, Tyr 844, and Lys 848 by Ala abolish CAR activity, indicating their key role in acid reduction. These results may assist in the functional annotation of CAR coding genes in genomic databases. While some other conserved residues decreased activity or had no significant impact, four residues increased the specific activity of NcCAR variants when replaced by alanine. Finally, we showed that NcCAR wild-type and mutants efficiently reduce aliphatic acids.

12.
Angew Chem Int Ed Engl ; 56(44): 13893-13897, 2017 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-28857436

RESUMO

The utilization of CO2 as a carbon source for organic synthesis meets the urgent demand for more sustainability in the production of chemicals. Herein, we report on the enzyme-catalyzed para-carboxylation of catechols, employing 3,4-dihydroxybenzoic acid decarboxylases (AroY) that belong to the UbiD enzyme family. Crystal structures and accompanying solution data confirmed that AroY utilizes the recently discovered prenylated FMN (prFMN) cofactor, and requires oxidative maturation to form the catalytically competent prFMNiminium species. This study reports on the in vitro reconstitution and activation of a prFMN-dependent enzyme that is capable of directly carboxylating aromatic catechol substrates under ambient conditions. A reaction mechanism for the reversible decarboxylation involving an intermediate with a single covalent bond between a quinoid adduct and cofactor is proposed, which is distinct from the mechanism of prFMN-associated 1,3-dipolar cycloadditions in related enzymes.

13.
Sci Rep ; 7: 46738, 2017 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-28466867

RESUMO

Homology and similarity based approaches are most widely used for the identification of new enzymes for biocatalysis. However, they are not suitable to find truly novel scaffolds with a desired function and this averts options and diversity. Hydroxynitrile lyases (HNLs) are an example of non-homologous isofunctional enzymes for the synthesis of chiral cyanohydrins. Due to their convergent evolution, finding new representatives is challenging. Here we show the discovery of unique HNL enzymes from the fern Davallia tyermannii by coalescence of transcriptomics, proteomics and enzymatic screening. It is the first protein with a Bet v1-like protein fold exhibiting HNL activity, and has a new catalytic center, as shown by protein crystallography. Biochemical properties of D. tyermannii HNLs open perspectives for the development of a complementary class of biocatalysts for the stereoselective synthesis of cyanohydrins. This work shows that systematic integration of -omics data facilitates discovery of enzymes with unpredictable sequences and helps to extend our knowledge about enzyme diversity.


Assuntos
Aldeído Liases/metabolismo , Antígenos de Plantas/metabolismo , Gleiquênias/enzimologia , Nitrilas/metabolismo , Proteínas de Plantas/metabolismo , Aldeído Liases/química , Aldeído Liases/genética , Sequência de Aminoácidos , Antígenos de Plantas/química , Antígenos de Plantas/genética , Sequência de Bases , Biocatálise , Cristalografia por Raios X , Gleiquênias/genética , Perfilação da Expressão Gênica/métodos , Modelos Moleculares , Nitrilas/química , Proteínas de Plantas/química , Proteínas de Plantas/genética , Conformação Proteica , Multimerização Proteica , Proteômica/métodos , Homologia de Sequência de Aminoácidos , Estereoisomerismo
15.
Angew Chem Int Ed Engl ; 56(26): 7615-7619, 2017 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-28544673

RESUMO

The Friedel-Crafts acylation is commonly used for the synthesis of aryl ketones, and a biocatalytic version, which may benefit from the chemo- and regioselectivity of enzymes, has not yet been introduced. Described here is a bacterial acyltransferase which can catalyze Friedel-Crafts C-acylation of phenolic substrates in buffer without the need of CoA-activated reagents. Conversions reach up to >99 %, and various C- or O-acyl donors, such as DAPG or isopropenyl acetate, are accepted by this enzyme. Furthermore the enzyme enables a Fries rearrangement-like reaction of resorcinol derivatives. These findings open an avenue for the development of alternative and selective C-C bond formation methods.


Assuntos
Biocatálise , Acilação , Aciltransferases/metabolismo , Catálise , DNA Bacteriano/genética , Escherichia coli/crescimento & desenvolvimento , Cinética , Fenóis/metabolismo , Pseudomonas/enzimologia , Pseudomonas/genética , Especificidade por Substrato
16.
PLoS One ; 12(2): e0171056, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28152088

RESUMO

Friedel-Crafts alkylation of aromatic systems is a classic reaction in organic chemistry, for which regiospecific mono-alkylation, however, is generally difficult to achieve. In nature, methyltransferases catalyze the addition of methyl groups to a wide range of biomolecules thereby modulating the physico-chemical properties of these compounds. Specifically, S-adenosyl-L-methionine dependent C-methyltransferases possess a high potential to serve as biocatalysts in environmentally benign organic syntheses. Here, we report on the high resolution crystal structure of CouO, a C-methyltransferase from Streptomyces rishiriensis involved in the biosynthesis of the antibiotic coumermycin A1. Through molecular docking calculations, site-directed mutagenesis and the comparison with homologous enzymes we identified His120 and Arg121 as key functional residues for the enzymatic activity of this group of C-methyltransferases. The elucidation of the atomic structure and the insight into the catalytic mechanism provide the basis for the (semi)-rational engineering of the enzyme in order to increase the substrate scope as well as to facilitate the acceptance of SAM-analogues as alternative cofactors.


Assuntos
Metiltransferases/química , Metiltransferases/metabolismo , Streptomyces/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Metiltransferases/genética , Modelos Moleculares , Simulação de Acoplamento Molecular , Conformação Proteica , S-Adenosil-Homocisteína/química , S-Adenosil-Homocisteína/metabolismo
17.
Sci Rep ; 6: 38183, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27905516

RESUMO

Transaminases are useful biocatalysts for the production of amino acids and chiral amines as intermediates for a broad range of drugs and fine chemicals. Here, we describe the discovery and characterisation of new transaminases from microorganisms which were enriched in selective media containing (R)-amines as sole nitrogen source. While most of the candidate proteins were clearly assigned to known subgroups of the fold IV family of PLP-dependent enzymes by sequence analysis and characterisation of their substrate specificity, some of them did not fit to any of these groups. The structure of one of these enzymes from Curtobacterium pusillum, which can convert d-amino acids and various (R)-amines with high enantioselectivity, was solved at a resolution of 2.4 Å. It shows significant differences especially in the active site compared to other transaminases of the fold IV family and thus indicates the existence of a new subgroup within this family. Although the discovered transaminases were not able to convert ketones in a reasonable time frame, overall, the enrichment-based approach was successful, as we identified two amine transaminases, which convert (R)-amines with high enantioselectivity, and can be used for a kinetic resolution of 1-phenylethylamine and analogues to obtain the (S)-amines with e.e.s >99%.


Assuntos
Actinobacteria/enzimologia , Proteínas de Bactérias/química , Dobramento de Proteína , Transaminases/química , Cristalografia por Raios X , Especificidade por Substrato
18.
FEBS J ; 283(19): 3587-3603, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27486859

RESUMO

The human dimethylglycine dehydrogenase (hDMGDH) is a flavin adenine dinucleotide (FAD)- and tetrahydrofolate (THF)-dependent, mitochondrial matrix enzyme taking part in choline degradation, one-carbon metabolism and electron transfer to the respiratory chain. The rare natural variant H109R causes dimethylglycine dehydrogenase deficiency leading to increased blood and urinary dimethylglycine concentrations. A detailed biochemical and structural characterization of hDMGDH was thus far hampered by insufficient heterologous expression of the protein. In the present study, we report the development of an intracellular, heterologous expression system in Komagataella phaffii (formerly known as Pichia pastoris) providing the opportunity to determine kinetic parameters, spectroscopic properties, thermostability, and the redox potential of hDMGDH. Moreover, we have successfully crystallized the wild-type enzyme and determined the structure to 3.1-Å resolution. The structure-based analysis of our biochemical data provided new insights into the kinetic properties of the enzyme in particular with respect to oxygen reactivity. A comparative study with the H109R variant demonstrated that the variant suffers from decreased protein stability, cofactor saturation, and substrate affinity. DATABASE: Structural data are available in the PDB database under the accession number 5L46.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Dimetilglicina Desidrogenase/química , Dimetilglicina Desidrogenase/genética , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Mutação Puntual , Dimetilglicina Desidrogenase/isolamento & purificação , Dimetilglicina Desidrogenase/metabolismo , Humanos , Cinética , Proteínas Mitocondriais/isolamento & purificação , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Oxirredução , Domínios Proteicos , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
19.
J Biotechnol ; 235: 24-31, 2016 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-27067080

RESUMO

Hydroxynitrile lyases (HNLs) catalyze the asymmetric addition of HCN to aldehydes producing enantiomerically pure cyanohydrins. These enzymes can be heterologously expressed in large quantities making them interesting candidates for industrial applications. The HNLs from Rosaceae evolved from flavin dependent dehydrogenase/oxidase structures. Here we report the high resolution X-ray structure of the highly glycosylated Prunus amygdalus HNL isoenzyme5 (PaHNL5 V317A) expressed in Aspergillus niger and its complex with benzyl alcohol. A comparison with the structure of isoenzyme PaHNL1 indicates a higher accessibility to the active site and a larger cavity for PaHNL5. Additionally, the PaHNL5 complex structure with benzyl alcohol was compared with the structurally related aryl-alcohol oxidase (AAO). Even though both enzymes contain an FAD-cofactor and histidine residues at crucial positions in the active site, PaHNL5 lacks the oxidoreductase activity. The structures indicate that in PaHNLs benzyl alcohol is bound too far away from the FAD cofactor in order to be oxidized.


Assuntos
Aldeído Liases , Flavinas/metabolismo , Proteínas de Plantas , Prunus dulcis/enzimologia , Aldeído Liases/química , Aldeído Liases/metabolismo , Aldeído Liases/ultraestrutura , Isoenzimas/química , Isoenzimas/metabolismo , Isoenzimas/ultraestrutura , Modelos Moleculares , Oxirredução , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/ultraestrutura
20.
Sci Rep ; 6: 23787, 2016 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-27025154

RESUMO

Human dipeptidyl-peptidase III (hDPP III) is a zinc-dependent hydrolase cleaving dipeptides off the N-termini of various bioactive peptides. Thus, the enzyme is likely involved in a number of physiological processes such as nociception and is also implicated in several forms of cancer. We present high-resolution crystal structures of hDPP III in complex with opioid peptides (Met-and Leu-enkephalin, endomorphin-2) as well as with angiotensin-II and the peptide inhibitor IVYPW. These structures confirm the previously reported large conformational change of the enzyme upon ligand binding and show that the structure of the closed conformation is independent of the nature of the bound peptide. The overall peptide-binding mode is also conserved ensuring the correct positioning of the scissile peptide bond with respect to the catalytic zinc ion. The structure of the angiotensin-II complex shows, how longer peptides are accommodated in the binding cleft of hDPP III. Differences in the binding modes allow a distinction between real substrates and inhibitory peptides or "slow" substrates. The latter displace a zinc bound water molecule necessitating the energetically much less favoured anhydride mechanism as opposed to the favoured promoted-water mechanism. The structural data also form the necessary framework for the design of specific hDPP III inhibitors.


Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/química , Encefalinas/química , Angiotensina II/química , Domínio Catalítico , Cristalografia por Raios X , Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Humanos , Ligação de Hidrogênio , Hidrólise , Cinética , Modelos Moleculares , Peptídeos Opioides/química , Ligação Proteica
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