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1.
Biochim Biophys Acta Proteins Proteom ; 1872(4): 141015, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38615986

RESUMO

The bifunctional enzyme, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC) is involved in catalyzing penultimate and final steps of purine de novo biosynthetic pathway crucial for the survival of organisms. The present study reports the characterization of ATIC from Candidatus Liberibacer asiaticus (CLasATIC) along with the identification of potential inhibitor molecules and evaluation of cell proliferative activity. CLasATIC showed both the AICAR Transformylase (AICAR TFase) activity for substrates, 10-f-THF (Km, 146.6 µM and Vmax, 0.95 µmol/min/mg) and AICAR (Km, 34.81 µM and Vmax, 0.56 µmol/min/mg) and IMP cyclohydrolase (IMPCHase) activitiy (Km, 1.81 µM and Vmax, 2.87 µmol/min/mg). The optimum pH and temperature were also identified for the enzyme activity. In-silico study has been conducted to identify potential inhibitor molecules through virtual screening and MD simulations. Out of many compounds, HNBSA, diosbulbin A and lepidine D emerged as lead compounds, exhibiting higher binding energy and stability for CLasATIC than AICAR. ITC study reports higher binding affinities for HNBSA and diosbulbin A (Kd, 12.3 µM and 34.2 µM, respectively) compared to AICAR (Kd, 83.4 µM). Likewise, DSC studies showed enhanced thermal stability for CLasATIC in the presence of inhibitors. CD and Fluorescence studies revealed significant conformational changes in CLasATIC upon binding of the inhibitors. CLasATIC demonstrated potent cell proliferative, wound healing and ROS scavenging properties evaluated by cell-based bioassays using CHO cells. This study highlights CLasATIC as a promising drug target with potential inhibitors for managing CLas and its unique cell protective, wound-healing properties for future biotechnological applications.


Assuntos
Aminoimidazol Carboxamida , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/química , Aminoimidazol Carboxamida/metabolismo , Aminoimidazol Carboxamida/farmacologia , Fosforribosilaminoimidazolcarboxamida Formiltransferase/metabolismo , Fosforribosilaminoimidazolcarboxamida Formiltransferase/química , Simulação de Acoplamento Molecular , Ribonucleotídeos/metabolismo , Ribonucleotídeos/química , Cinética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/antagonistas & inibidores , Nucleotídeo Desaminases/metabolismo , Nucleotídeo Desaminases/química , Nucleotídeo Desaminases/genética , Especificidade por Substrato , Proliferação de Células/efeitos dos fármacos , Hidroximetil e Formil Transferases/metabolismo , Hidroximetil e Formil Transferases/química , Hidroximetil e Formil Transferases/genética , Hidroximetil e Formil Transferases/antagonistas & inibidores , Complexos Multienzimáticos
2.
J Am Chem Soc ; 142(46): 19754-19762, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33147013

RESUMO

The congested nature of quaternary carbons hinders their preparation, most notably when stereocontrol is required. Here we report a biocatalytic method for the creation of quaternary carbon centers with broad substrate scope, leading to different compound classes bearing this structural feature. The key step comprises the aldol addition of 3,3-disubstituted 2-oxoacids to aldehydes catalyzed by metal dependent 3-methyl-2-oxobutanoate hydroxymethyltransferase from E. coli (KPHMT) and variants thereof. The 3,3,3-trisubstituted 2-oxoacids thus produced were converted into 2-oxolactones and 3-hydroxy acids and directly to ulosonic acid derivatives, all bearing gem-dialkyl, gem-cycloalkyl, and spirocyclic quaternary centers. In addition, some of these reactions use a single enantiomer from racemic nucleophiles to afford stereopure quaternary carbons. The notable substrate tolerance and stereocontrol of these enzymes are indicative of their potential for the synthesis of structurally intricate molecules.


Assuntos
Aldeídos/metabolismo , Proteínas de Escherichia coli/metabolismo , Hidroximetil e Formil Transferases/metabolismo , Cetoácidos/metabolismo , Aldeídos/química , Sítios de Ligação , Biocatálise , Domínio Catalítico , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Hidroximetil e Formil Transferases/química , Hidroximetil e Formil Transferases/genética , Cetoácidos/química , Mutagênese Sítio-Dirigida , Estereoisomerismo , Especificidade por Substrato
3.
Emerg Microbes Infect ; 9(1): 58-66, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31894729

RESUMO

Enzymes from the purine salvage pathway in Mycobacterium tuberculosis (Mtb) have been regarded as an attractive target for the development of anti-bacterial drugs. Although this pathway has not been extensively studied in Mtb, it has been identified as essential for growth and survival. Glycinamide-RNase-transformylase T (PurT) is found only in some specific bacteria including Mtb and utilizes ATP-dependent ligation to catalyze the formylation of 5'-phosphoribosyl-glycinamide (GAR) in the third reaction of the de novo purine salvage pathway. In the study, we determined the crystal structure of MtbPurT at a resolution of 2.79 Å. In contrast to Pyrococcus horikoshii OT3 PurT (phBCCPPurT), MtbPurT exhibits an "open" conformation, which results in a broader ATP-binding pocket and thus might facilitate the entry and exit of the cofactor. Additionally, active site superposition with E.coli PurT (EcPurT) showed that residues involved in the ATP-binding site in MtbPurT exhibited structural similarity but had notable difference in the GAR-binding site. The loop 383-389 in MtbPurT was much shorter and shifted 5.7 Å away from the phosphate of the GAR substrate. The different GAR-binding mode might result in a large conformational change in MtbPurT, and would provide a possible opportunity for anti-TB drug development.


Assuntos
Proteínas de Bactérias/química , Hidroximetil e Formil Transferases/química , Mycobacterium tuberculosis/enzimologia , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Hidroximetil e Formil Transferases/metabolismo , Redes e Vias Metabólicas , Purinas/metabolismo
4.
Protein Sci ; 29(4): 930-940, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31867814

RESUMO

Tens of thousands of bacterial genome sequences are now known due to the development of rapid and inexpensive sequencing technologies. An important key in utilizing these vast amounts of data in a biologically meaningful way is to infer the function of the proteins encoded in the genomes via bioinformatics techniques. Whereas these approaches are absolutely critical to the annotation of gene function, there are still issues of misidentifications, which must be experimentally corrected. For example, many of the bacterial DNA sequences encoding sugar N-formyltransferases have been annotated as l-methionyl-tRNA transferases in the databases. These mistakes may be due in part to the fact that until recently the structures and functions of these enzymes were not well known. Herein we describe the misannotation of two genes, WP_088211966.1 and WP_096244125.1, from Shewanella spp. and Pseudomonas congelans, respectively. Although the proteins encoded by these genes were originally suggested to function as l-methionyl-tRNA transferases, we demonstrate that they actually catalyze the conversion of dTDP-4-amino-4,6-dideoxy-d-glucose to dTDP-4-formamido-4,6-dideoxy-d-glucose utilizing N10 -formyltetrahydrofolate as the carbon source. For this analysis, the genes encoding these enzymes were cloned and the corresponding proteins purified. X-ray structures of the two proteins were determined to high resolution and kinetic analyses were conducted. Both enzymes display classical Michaelis-Menten kinetics and adopt the characteristic three-dimensional structural fold previously observed for other sugar N-formyltransferases. The results presented herein will aid in the future annotation of these fascinating enzymes.


Assuntos
Hidroximetil e Formil Transferases/genética , Pseudomonas/enzimologia , Shewanella/enzimologia , Açúcares/metabolismo , Configuração de Carboidratos , Cristalografia por Raios X , Hidroximetil e Formil Transferases/química , Hidroximetil e Formil Transferases/metabolismo , Cinética , Modelos Moleculares , Açúcares/química
5.
Sci Rep ; 9(1): 16316, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31705139

RESUMO

The hydroxymethylation of cytosine bases plays a vital role in the phage DNA protection system inside the host Escherichia coli. This modification is known to be catalyzed by the dCMP hydroxymethylase from bacteriophage T4 (T4dCH); structural information on the complexes with the substrate, dCMP and the co-factor, tetrahydrofolate is currently available. However, the detailed mechanism has not been understood clearly owing to a lack of structure in the complex with a reaction intermediate. We have applied the X-ray free electron laser (XFEL) technique to determine a high-resolution structure of a T4dCH D179N active site mutant. The XFEL structure was determined at room temperature and exhibited several unique features in comparison with previously determined structures. Unexpectedly, we observed a bulky electron density at the active site of the mutant that originated from the physiological host (i.e., E. coli). Mass-spectrometric analysis and a cautious interpretation of an electron density map indicated that it was a dTMP molecule. The bound dTMP mimicked the methylene intermediate from dCMP to 5'-hydroxymethy-dCMP, and a critical water molecule for the final hydroxylation was convincingly identified. Therefore, this study provides information that contributes to the understanding of hydroxymethylation.


Assuntos
Bacteriófago T4/enzimologia , Elétrons , Hidroximetil e Formil Transferases/química , Hidroximetil e Formil Transferases/genética , Lasers , Mutação , Timidina Monofosfato/metabolismo , Cristalografia por Raios X , Hidroximetil e Formil Transferases/metabolismo , Modelos Moleculares , Conformação Proteica , Água/química
6.
Proc Natl Acad Sci U S A ; 116(51): 25583-25590, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31776258

RESUMO

Methylotrophy, the ability of microorganisms to grow on reduced one-carbon substrates such as methane or methanol, is a feature of various bacterial species. The prevailing oxidation pathway depends on tetrahydromethanopterin (H4MPT) and methylofuran (MYFR), an analog of methanofuran from methanogenic archaea. Formyltransferase/hydrolase complex (Fhc) generates formate from formyl-H4MPT in two consecutive reactions where MYFR acts as a carrier of one-carbon units. Recently, we chemically characterized MYFR from the model methylotroph Methylorubrum extorquens and identified an unusually long polyglutamate side chain of up to 24 glutamates. Here, we report on the crystal structure of Fhc to investigate the function of the polyglutamate side chain in MYFR and the relatedness of the enzyme complex with the orthologous enzymes in archaea. We identified MYFR as a prosthetic group that is tightly, but noncovalently, bound to Fhc. Surprisingly, the structure of Fhc together with MYFR revealed that the polyglutamate side chain of MYFR is branched and contains glutamates with amide bonds at both their α- and γ-carboxyl groups. This negatively charged and branched polyglutamate side chain interacts with a cluster of conserved positively charged residues of Fhc, allowing for strong interactions. The MYFR binding site is located equidistantly from the active site of the formyltransferase (FhcD) and metallo-hydrolase (FhcA). The polyglutamate serves therefore an additional function as a swinging linker to shuttle the one-carbon carrying amine between the two active sites, thereby likely increasing overall catalysis while decreasing the need for high intracellular MYFR concentrations.


Assuntos
Proteínas de Bactérias , Furanos , Hidroximetil e Formil Transferases , Metano , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Coenzimas/química , Coenzimas/metabolismo , Cristalografia , Formiatos/química , Formiatos/metabolismo , Furanos/química , Furanos/metabolismo , Hidroximetil e Formil Transferases/química , Hidroximetil e Formil Transferases/genética , Hidroximetil e Formil Transferases/metabolismo , Metano/química , Metano/metabolismo , Metanol/química , Metanol/metabolismo , Methylobacterium extorquens/enzimologia , Methylobacterium extorquens/genética , Ácido Poliglutâmico/química , Ácido Poliglutâmico/metabolismo
7.
Protein Sci ; 28(4): 707-716, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30666752

RESUMO

Pantoea ananatis is a Gram-negative bacterium first recognized in 1928 as the causative agent of pineapple rot in the Philippines. Since then various strains of the organism have been implicated in the devastation of agriculturally important crops. Some strains, however, have been shown to function as non-pathogenic plant growth promoting organisms. To date, the factors that determine pathogenicity or lack thereof between the various strains are not well understood. All P. ananatis strains contain lipopolysaccharides, which differ with respect to the identities of their associated sugars. Given our research interest on the presence of the unusual sugar, 4-formamido-4,6-dideoxy-d-glucose, found on the lipopolysaccharides of Campylobacter jejuni and Francisella tularensis, we were curious as to whether other bacteria have the appropriate biosynthetic machinery to produce these unique carbohydrates. Four enzymes are typically required for their biosynthesis: a thymidylyltransferase, a 4,6-dehydratase, an aminotransferase, and an N-formyltransferase. Here, we report that the gene SAMN03097714_1080 from the P. ananatis strain NFR11 does, indeed, encode for an N-formyltransferase, hereafter referred to as PA1080c. Our kinetic analysis demonstrates that PA1080c displays classical Michaelis-Menten kinetics with dTDP-4-amino-4,6-dideoxy-d-glucose as the substrate and N10 -formyltetrahydrofolate as the carbon source. In addition, the X-ray structure of PA1080c, determined to 1.7 Å resolution, shows that the enzyme adopts the molecular architecture observed for other sugar N-formyltransferases. Analysis of the P. ananatis NFR11 genome suggests that the three other enzymes necessary for N-formylated sugar biosynthesis are also present. Intriguingly, those strains of P. ananatis that are non-pathogenic apparently do not contain these genes.


Assuntos
Hidroximetil e Formil Transferases/metabolismo , Pantoea/metabolismo , Proteínas de Plantas/metabolismo , Vias Biossintéticas , Cristalografia por Raios X , Hidroximetil e Formil Transferases/química , Lipopolissacarídeos/metabolismo , Modelos Moleculares , Pantoea/química , Doenças das Plantas/microbiologia , Proteínas de Plantas/química , Plantas/microbiologia , Conformação Proteica , Especificidade por Substrato , Açúcares/metabolismo
8.
Arch Biochem Biophys ; 664: 40-50, 2019 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-30689984

RESUMO

The hydroxyornithine transformylase from Pseudomonas aeruginosa is known by the gene name pvdF, and has been hypothesized to use N10-formyltetrahydrofolate (N10-fTHF) as a co-substrate formyl donor to convert N5-hydroxyornithine (OHOrn) to N5-formyl- N5-hydroxyornithine (fOHOrn). PvdF is in the biosynthetic pathway for pyoverdin biosynthesis, a siderophore generated under iron-limiting conditions that has been linked to virulence, quorum sensing and biofilm formation. The structure of PvdF was determined by X-ray crystallography to 2.3 Å, revealing a formyltransferase fold consistent with N10-formyltetrahydrofolate dependent enzymes, such as the glycinamide ribonucleotide transformylases, N-sugar transformylases and methionyl-tRNA transformylases. Whereas the core structure, including the catalytic triad, is conserved, PvdF has three insertions of 18 or more amino acids, which we hypothesize are key to binding the OHOrn substrate. Steady state kinetics revealed a non-hyperbolic rate curve, promoting the hypothesis that PvdF uses a random-sequential mechanism, and favors folate binding over OHOrn.


Assuntos
Formiltetra-Hidrofolatos/metabolismo , Hidroximetil e Formil Transferases/química , Hidroximetil e Formil Transferases/metabolismo , Oligopeptídeos/biossíntese , Ácido Fólico/metabolismo , Modelos Moleculares , Conformação Proteica , Pseudomonas aeruginosa/enzimologia
9.
ACS Chem Biol ; 13(11): 3161-3172, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30346688

RESUMO

Nonribosomal peptide synthetases (NRPSs) increase the chemical diversity of their products by acquiring tailoring domains. Linear gramicidin synthetase starts with a tailoring formylation (F) domain, which likely originated from a sugar formyltransferase (FT) gene. Here, we present studies on an Anoxybacillus kamchatkensis sugar FT representative of the prehorizontal gene transfer FT. Gene cluster analysis reveals that this FT acts on a UDP-sugar in a novel pathway for synthesis of a 7-formamido derivative of CMP-pseudaminic acid. We recapitulate the pathway up to and including the formylation step in vitro, experimentally demonstrating the role of the FT. We also present X-ray crystal structures of the FT alone and with ligands, which unveil contrasts with other structurally characterized sugar FTs and show close structural similarity with the F domain. The structures reveal insights into the adaptations that were needed to co-opt and evolve a sugar FT into a functional and useful NRPS domain.


Assuntos
Hidroximetil e Formil Transferases/química , Peptídeo Sintases/química , Anoxybacillus/enzimologia , Cristalografia por Raios X , Transferência Genética Horizontal , Hidroliases/química , Hidroliases/genética , Hidroximetil e Formil Transferases/genética , Ligantes , Família Multigênica , Peptídeo Sintases/genética , Domínios Proteicos , Transaminases/química , Transaminases/genética
10.
Biochim Biophys Acta Proteins Proteom ; 1866(2): 254-263, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29042184

RESUMO

Aminoimidazolecarboxamide ribonucleotide formyl transferase (AICARFT): Inosine monophosphate cyclohydrolase (IMPCH, collectively called ATIC) is a bifunctional enzyme that catalyses the penultimate and final steps in the purine de novo biosynthesis pathway. The bifunctional protein is dimeric and each monomer contains two different active sites both of which are capable of binding nucleotide substrates, this means to a potential total of four distinct binding events might be observed. Within this work we used a combination of site-directed and truncation mutants of ATIC to independently investigate the binding at these two sites using calorimetry. A single S10W mutation is sufficient to block the IMPCH active site allowing investigation of the effects of mutation on ligand binding in the AICARFT active site. The majority of nucleotide ligands bind selectively at one of the two active sites with the exception of xanthosine monophosphate, XMP, which, in addition to binding in both AICARFT and IMPCH active sites, shows evidence for cooperative binding with communication between symmetrically-related active sites in the two IMPCH domains. The AICARFT site is capable of independently binding both nucleotide and folate substrates with high affinity however no evidence for positive cooperativity in binding could be detected using the model ligands employed in this study.


Assuntos
Hidroximetil e Formil Transferases/química , Modelos Moleculares , Complexos Multienzimáticos/química , Nucleotídeo Desaminases/química , Nucleotídeos/química , Domínio Catalítico , Humanos , Hidroximetil e Formil Transferases/genética , Hidroximetil e Formil Transferases/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Nucleotídeo Desaminases/genética , Nucleotídeo Desaminases/metabolismo , Nucleotídeos/genética , Nucleotídeos/metabolismo , Ligação Proteica , Especificidade por Substrato/fisiologia
11.
FEBS J ; 284(24): 4233-4261, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29063699

RESUMO

The 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC) catalyzes final two steps of purine nucleotide de novo biosynthetic pathway. This study reports the characterization of ATIC from Staphylococcus lugdunensis (SlugATIC). Apart from kinetic analysis and a detailed biophysical characterization of SlugATIC, the role of ATIC in cell proliferation has been demonstrated for the first time. The purified recombinant SlugATIC and its truncated domains exist mainly in dimeric form was revealed in gel-filtration and glutaraldehyde cross-linking studies. The two activities reside on separate domains was demonstrated in kinetic analysis of SlugATIC and reconstituted truncated N-terminal IMP cyclohydrolase (IMPCHase) and C-terminal AICAR transformylase (AICAR TFase) domains. Site-directed mutagenesis showed that Lys255 and His256 are the key catalytic residues, while Asn415 substantially contributes to AICAR TFase activity in SlugATIC. The differential scanning calorimetry (DSC) analysis revealed a molten globule-like structure for independent N-terminal domain as compared with a relatively stable conformational state in full-length SlugATIC signifying the importance of covalently linked domains. Unlike reported crystal structures, the DSC studies revealed significant conformational changes on binding of leading ligand to AICAR TFase domain in SlugATIC. The cell proliferation activity of SlugATIC was observed where it promoted proliferation and viability of NIH 3T3 and RIN-5F cells, exhibited in vitro wound healing in NIH 3T3 fibroblast cells, and rescued RIN-5F cells from the cytotoxic effects of palmitic acid and high glucose. The results suggest that ATIC, an important drug target, can also be exploited for its cell proliferative properties.


Assuntos
Proteínas de Bactérias/fisiologia , Hidroximetil e Formil Transferases/fisiologia , Complexos Multienzimáticos/fisiologia , Nucleotídeo Desaminases/fisiologia , Staphylococcus lugdunensis/enzimologia , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Varredura Diferencial de Calorimetria , Divisão Celular/efeitos dos fármacos , Glucose/toxicidade , Hidroximetil e Formil Transferases/química , Hidroximetil e Formil Transferases/genética , Hidroximetil e Formil Transferases/isolamento & purificação , Inosina Monofosfato/farmacologia , Camundongos , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/isolamento & purificação , Mutação , Células NIH 3T3 , Nucleotídeo Desaminases/química , Nucleotídeo Desaminases/genética , Nucleotídeo Desaminases/isolamento & purificação , Ácido Palmítico/toxicidade , Conformação Proteica , Domínios Proteicos , Ratos , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleotídeos/farmacologia , Staphylococcus lugdunensis/genética , Cicatrização/efeitos dos fármacos
12.
Biochemistry ; 56(29): 3818-3825, 2017 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-28665588

RESUMO

The causative agent of tuberculosis, Mycobacterium tuberculosis, is a bacterium with a complex cell wall and a complicated life cycle. The genome of M. tuberculosis contains well over 4000 genes thought to encode proteins. One of these codes for a putative enzyme referred to as Rv3404c, which has attracted research attention as a potential virulence factor for over 12 years. Here we demonstrate that Rv3404c functions as a sugar N-formyltransferase that converts dTDP-4-amino-4,6-dideoxyglucose into dTDP-4-formamido-4,6-dideoxyglucose using N10-formyltetrahydrofolate as the carbon source. Kinetic analyses demonstrate that Rv3404c displays a significant catalytic efficiency of 1.1 × 104 M-1 s-1. In addition, we report the X-ray structure of a ternary complex of Rv3404c solved in the presence of N5-formyltetrahydrofolate and dTDP-4-amino-4,6-dideoxyglucose. The final model of Rv3404c was refined to an overall R-factor of 16.8% at 1.6 Å resolution. The results described herein are especially intriguing given that there have been no published reports of N-formylated sugars associated with M. tuberculosis. The data thus provide a new avenue of research into this fascinating, yet deadly, organism that apparently has been associated with human infection since ancient times.


Assuntos
Proteínas de Bactérias/química , Hidroximetil e Formil Transferases/química , Modelos Moleculares , Mycobacterium tuberculosis/enzimologia , Fatores de Virulência/química , Proteínas de Bactérias/metabolismo , Catálise , Cristalografia por Raios X , Desoxiaçúcares/química , Desoxiaçúcares/metabolismo , Formiltetra-Hidrofolatos/química , Formiltetra-Hidrofolatos/metabolismo , Hidroximetil e Formil Transferases/metabolismo , Cinética , Mycobacterium tuberculosis/patogenicidade , Nucleotídeos de Timina/química , Nucleotídeos de Timina/metabolismo , Fatores de Virulência/metabolismo
13.
Biochemistry ; 56(28): 3657-3668, 2017 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-28636341

RESUMO

It has become increasingly apparent within the last several years that unusual N-formylated sugars are often found on the O-antigens of such Gram negative pathogenic organisms as Francisella tularensis, Campylobacter jejuni, and Providencia alcalifaciens, among others. Indeed, in some species of Brucella, for example, the O-antigen contains 1,2-linked 4-formamido-4,6-dideoxy-α-d-mannosyl groups. These sugars, often referred to as N-formylperosamine, are synthesized in pathways initiating with GDP-mannose. One of the enzymes required for the production of N-formylperosamine, namely, WbkC, was first identified in 2000 and was suggested to function as an N-formyltransferase. Its biochemical activity was never experimentally verified, however. Here we describe a combined structural and functional investigation of WbkC from Brucella melitensis. Four high resolution X-ray structures of WbkC were determined in various complexes to address its active site architecture. Unexpectedly, the quaternary structure of WbkC was shown to be different from that previously observed for other sugar N-formyltransferases. Additionally, the structures revealed a second binding site for a GDP molecule distinct from that required for GDP-perosamine positioning. In keeping with this additional binding site, kinetic data with the wild type enzyme revealed complex patterns. Removal of GDP binding by mutating Phe 142 to an alanine residue resulted in an enzyme variant displaying normal Michaelis-Menten kinetics. These data suggest that this nucleotide binding pocket plays a role in enzyme regulation. Finally, by using an alternative substrate, we demonstrate that WbkC can be utilized to produce a trideoxysugar not found in nature.


Assuntos
Brucella melitensis/enzimologia , Hidroximetil e Formil Transferases/metabolismo , Sítios de Ligação , Brucella melitensis/química , Brucelose/microbiologia , Domínio Catalítico , Cristalografia por Raios X , Guanosina Difosfato/metabolismo , Hexosaminas/metabolismo , Humanos , Hidroximetil e Formil Transferases/química , Cinética , Modelos Moleculares , Conformação Proteica , Especificidade por Substrato
14.
J Struct Biol ; 200(3): 267-278, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28263875

RESUMO

N-formylated sugars are found on the lipopolysaccharides of various pathogenic Gram negative bacteria including Campylobacter jejuni 81116, Francisella tularensis, Providencia alcalifaciens O30, and Providencia alcalifaciens O40. The last step in the biosynthetic pathways for these unusual sugars is catalyzed by N-formyltransferases that utilize N10-formyltetrahydrofolate as the carbon source. The substrates are dTDP-linked amino sugars with the functional groups installed at either the C-3' or C-4' positions of the pyranosyl rings. Here we describe a structural and enzymological investigation of the putative N-formyltransferase, FdtF, from Salmonella enterica O60. In keeping with its proposed role in the organism, the kinetic data reveal that the enzyme is more active with dTDP-3-amino-3,6-dideoxy-d-galactose than with dTDP-3-amino-3,6-dideoxy-d-glucose. The structural data demonstrate that the enzyme contains, in addition to the canonical N-formyltransferase fold, an ankyrin repeat moiety that houses a second dTDP-sugar binding pocket. This is only the second time an ankyrin repeat has been shown to be involved in small molecule binding. The research described herein represents the first structural analysis of a sugar N-formyltransferase that specifically functions on dTDP-3-amino-3,6-dideoxy-d-galactose in vivo and thus adds to our understanding of these intriguing enzymes.


Assuntos
Proteínas de Bactérias/química , Hidroximetil e Formil Transferases/química , Hidroximetil e Formil Transferases/metabolismo , Salmonella enterica/enzimologia , Amino Açúcares/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Glucosamina/análogos & derivados , Glucosamina/metabolismo , Hidroximetil e Formil Transferases/genética , Cinética , Modelos Moleculares , Conformação Proteica
15.
BMC Struct Biol ; 17(1): 1, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28143508

RESUMO

BACKGROUND: The post-translational modification pathway referred to as pupylation marks proteins for proteasomal degradation in Mycobacterium tuberculosis and other actinobacteria by covalently attaching the small protein Pup (prokaryotic ubiquitin-like protein) to target lysine residues. In contrast to the functionally analogous eukaryotic ubiquitin, Pup is intrinsically disordered in its free form. Its unfolded state allows Pup to adopt different structures upon interaction with different binding partners like the Pup ligase PafA and the proteasomal ATPase Mpa. While the disordered behavior of free Pup has been well characterized, it remained unknown whether Pup adopts a distinct structure when attached to a substrate. RESULTS: Using a combination of NMR experiments and biochemical analysis we demonstrate that Pup remains unstructured when ligated to two well-established pupylation substrates targeted for proteasomal degradation in Mycobacterium tuberculosis, malonyl transacylase (FabD) and ketopantoyl hydroxylmethyltransferase (PanB). Isotopically labeled Pup was linked to FabD and PanB by in vitro pupylation to generate homogeneously pupylated substrates suitable for NMR analysis. The single target lysine of PanB was identified by a combination of mass spectroscopy and mutational analysis. Chemical shift comparison between Pup in its free form and ligated to substrate reveals intrinsic disorder of Pup in the conjugate. CONCLUSION: When linked to the proteasomal substrates FabD and PanB, Pup is unstructured and retains the ability to interact with its different binding partners. This suggests that it is not the conformation of Pup attached to these two substrates which determines their delivery to the proteasome, but the availability of the degradation complex and the depupylase.


Assuntos
Proteína de Transporte de Acila S-Maloniltransferase/química , Proteínas de Bactérias/química , Hidroximetil e Formil Transferases/química , Mycobacterium tuberculosis/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinas/química , Proteína de Transporte de Acila S-Maloniltransferase/metabolismo , Proteínas de Bactérias/metabolismo , Hidroximetil e Formil Transferases/metabolismo , Modelos Moleculares , Conformação Proteica , Proteólise , Especificidade por Substrato , Ubiquitinação , Ubiquitinas/metabolismo
16.
Curr Opin Struct Biol ; 41: 1-9, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27209114

RESUMO

The N-formyltransferases, also known as transformylases, play key roles in de novo purine biosynthesis where they catalyze the transfer of formyl groups to primary amine acceptors. These enzymes require N10-formyltetrahydrofolate for activity. Due to their biological importance they have been extensively investigated for many years, and they are still serving as targets for antifolate drug design. Most of our understanding of the N-formyltransferases has been derived from these previous studies. It is now becoming increasingly apparent, however, that N-formylation also occurs on some amino sugars found on the O-antigens of pathogenic bacteria. This review focuses on recent developments in the biochemical and structural characterization of the sugar N-formyltransferases.


Assuntos
Metabolismo dos Carboidratos , Carboidratos/biossíntese , Hidroximetil e Formil Transferases/metabolismo , Bactérias/enzimologia , Humanos , Hidroximetil e Formil Transferases/química
17.
Nature ; 529(7585): 239-42, 2016 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-26762462

RESUMO

Nonribosomal peptide synthetases (NRPSs) are very large proteins that produce small peptide molecules with wide-ranging biological activities, including environmentally friendly chemicals and many widely used therapeutics. NRPSs are macromolecular machines, with modular assembly-line logic, a complex catalytic cycle, moving parts and many active sites. In addition to the core domains required to link the substrates, they often include specialized tailoring domains, which introduce chemical modifications and allow the product to access a large expanse of chemical space. It is still unknown how the NRPS tailoring domains are structurally accommodated into megaenzymes or how they have adapted to function in nonribosomal peptide synthesis. Here we present a series of crystal structures of the initiation module of an antibiotic-producing NRPS, linear gramicidin synthetase. This module includes the specialized tailoring formylation domain, and states are captured that represent every major step of the assembly-line synthesis in the initiation module. The transitions between conformations are large in scale, with both the peptidyl carrier protein domain and the adenylation subdomain undergoing huge movements to transport substrate between distal active sites. The structures highlight the great versatility of NRPSs, as small domains repurpose and recycle their limited interfaces to interact with their various binding partners. Understanding tailoring domains is important if NRPSs are to be utilized in the production of novel therapeutics.


Assuntos
Biocatálise , Brevibacillus/enzimologia , Gramicidina/biossíntese , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Isomerases de Aminoácido/química , Isomerases de Aminoácido/metabolismo , Antibacterianos/biossíntese , Sítios de Ligação , Metabolismo dos Carboidratos , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Domínio Catalítico , Coenzimas/metabolismo , Cristalografia por Raios X , Hidroximetil e Formil Transferases/química , Hidroximetil e Formil Transferases/metabolismo , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Panteteína/análogos & derivados , Panteteína/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , RNA de Transferência/química , RNA de Transferência/metabolismo
18.
Biophys J ; 109(10): 2182-94, 2015 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-26588576

RESUMO

Subcellular compartmentalization of biomolecules and their reactions is common in biology and provides a general strategy for improving and/or controlling kinetics in metabolic pathways that contain multiple sequential enzymes. Enzymes can be colocalized in multiprotein complexes, on scaffolds or inside subcellular organelles. Liquid organelles formed by intracellular phase coexistence could provide an additional means of sequential enzyme colocalization. Here we use experiment and computation to explore the kinetic consequences of sequential enzyme compartmentalization into model liquid organelles in a crowded polymer solution. Two proteins of the de novo purine biosynthesis pathway, ASL (adenylosuccinate lyase, Step 8) and ATIC (5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase, Steps 9 and 10), were studied in a polyethylene glycol/dextran aqueous two-phase system. Dextran-rich phase droplets served as model liquid compartments for enzyme colocalization. In this system, which lacks any specific binding interactions between the phase-forming polymers and the enzymes, we did not observe significant rate enhancements from colocalization for the overall reaction under our experimental conditions. The experimental results were used to adapt a mathematical model to quantitatively describe the kinetics. The mathematical model was then used to explore additional, experimentally inaccessible conditions to predict when increased local concentrations of enzymes and substrates can (or cannot) be expected to yield increased rates of product formation. Our findings indicate that colocalization within these simplified model liquid organelles can lead to enhanced metabolic rates under some conditions, but that very strong partitioning into the phase that serves as the compartment is necessary. In vivo, this could be provided by specific binding affinities between components of the liquid compartment and the molecules to be localized within it.


Assuntos
Adenilossuccinato Liase/metabolismo , Compartimento Celular , Hidroximetil e Formil Transferases/metabolismo , Modelos Biológicos , Complexos Multienzimáticos/metabolismo , Nucleotídeo Desaminases/metabolismo , Adenilossuccinato Liase/química , Humanos , Hidroximetil e Formil Transferases/química , Lipossomos/química , Complexos Multienzimáticos/química , Nucleotídeo Desaminases/química
19.
Protein Sci ; 24(6): 976-86, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25752909

RESUMO

The existence of N-formylated sugars in the O-antigens of Gram-negative bacteria has been known since the middle 1980s, but only recently have the biosynthetic pathways for their production been reported. In these pathways, glucose-1-phosphate is first activated by attachment to a dTMP moiety. This step is followed by a dehydration reaction and an amination. The last step in these pathways is catalyzed by N-formyltransferases that utilize N(10) -formyltetrahydrofolate as the carbon source. Here we describe the three-dimensional structure of one of these N-formyltransferases, namely VioF from Providencia alcalifaciens O30. Specifically, this enzyme catalyzes the conversion of dTDP-4-amino-4,6-dideoxyglucose (dTDP-Qui4N) to dTDP-4,6-dideoxy-4-formamido-d-glucose (dTDP-Qui4NFo). For this analysis, the structure of VioF was solved to 1.9 Å resolution in both its apoform and in complex with tetrahydrofolate and dTDP-Qui4N. The crystals used in the investigation belonged to the space group R32 and demonstrated reticular merohedral twinning. The overall catalytic core of the VioF subunit is characterized by a six stranded mixed ß-sheet flanked on one side by three α-helices and on the other side by mostly random coil. This N-terminal domain is followed by an α-helix and a ß-hairpin that form the subunit:subunit interface. The active site of the enzyme is shallow and solvent-exposed. Notably, the pyranosyl moiety of dTDP-Qui4N is positioned into the active site by only one hydrogen bond provided by Lys 77. Comparison of the VioF model to that of a previously determined N-formyltransferase suggests that substrate specificity is determined by interactions between the protein and the pyrophosphoryl group of the dTDP-sugar substrate.


Assuntos
Proteínas de Bactérias/química , Hidroximetil e Formil Transferases/química , Providencia/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Formiltetra-Hidrofolatos/metabolismo , Hidroximetil e Formil Transferases/genética , Hidroximetil e Formil Transferases/metabolismo , Modelos Moleculares , Conformação Proteica , Providencia/genética
20.
Biochemistry ; 54(3): 631-8, 2015 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-25574689

RESUMO

N-Formylated sugars such as 3,6-dideoxy-3-formamido-d-glucose (Qui3NFo) have been observed on the lipopolysaccharides of various pathogenic bacteria, including Providencia alcalifaciens, a known cause of gastroenteritis. These unusual carbohydrates are synthesized in vivo as dTDP-linked sugars. The biosynthetic pathway for the production of dTDP-Qui3NFo requires five enzymes with the last step catalyzed by an N-formyltransferase that utilizes N(10)-tetrahydrofolate as a cofactor. Here we describe a structural and functional investigation of the P. alcalifaciens N-formyltransferase, hereafter referred to as QdtF. For this analysis, the structure of the dimeric enzyme was determined in the presence of N(5)-formyltetrahydrofolate, a stable cofactor, and dTDP-3,6-dideoxy-3-amino-d-glucose (dTDP-Qui3N) to 1.5 Å resolution. The overall fold of the subunit consists of three regions with the N-terminal and middle motifs followed by an ankyrin repeat domain. Whereas the ankyrin repeat is a common eukaryotic motif involved in protein-protein interactions, reports of its presence in prokaryotic enzymes have been limited. Unexpectedly, this ankyrin repeat houses a second binding pocket for dTDP-Qui3N, which is characterized by extensive interactions between the protein and the ligand. To address the effects of this second binding site on catalysis, a site-directed mutant protein, W305A, was constructed. Kinetic analyses demonstrated that the catalytic activity of the W305A variant was reduced by approximately 7-fold. The structure of the W305A mutant protein in complex with N(5)-formyltetrahydrofolate and dTDP-Qui3N was subsequently determined to 1.5 Å resolution. The electron density map clearly showed that ligand binding had been completely abolished in the auxiliary pocket. The wild-type enzyme was also tested for activity against dTDP-3,6-dideoxy-3-amino-d-galactose (dTDP-Fuc3N) as a substrate. Strikingly, sigmoidal kinetics indicating homotropic allosteric behavior were observed. Although the identity of the ligand that regulates QdtF activity in vivo is at present unknown, our results still provide the first example of an ankyrin repeat functioning in small molecule binding.


Assuntos
Repetição de Anquirina , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Hidroximetil e Formil Transferases/química , Hidroximetil e Formil Transferases/metabolismo , Providencia/enzimologia , Sítio Alostérico , Domínio Catalítico , Cinética , Ligantes , Modelos Moleculares , Eletricidade Estática , Relação Estrutura-Atividade , Especificidade por Substrato , Nucleotídeos de Timina/metabolismo
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