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1.
J Agric Food Chem ; 67(45): 12382-12392, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31635461

RESUMO

Protoporphyrinogen oxidase (PPO, EC 1.3.3.4) is a promising target for herbicide discovery. Search for new compounds with novel chemotypes is a key objective for agrochemists. Here, we describe the discovery and systematic SAR-based structure optimization of novel N-isoxazolinylphenyltriazinones 5-9 as PPO inhibitors. The in vivo herbicidal activity and in vitro Nicotiana tabacum PPO (NtPPO) inhibitory activity were explored in detail. A number of the new synthetic compounds displayed strong PPO inhibitory activity with Ki values in the nanomolar range. Some compounds exhibited excellent and broad-spectrum weed control at the rate of 9.375-37.5 g ai/ha by postemergence application and showed improved monocotyledonous weed control compared to saflufenacil. Most promisingly, ethyl 3-(2-chloro-5-(3,5-dimethyl-2,6-dioxo-4-thioxo-1,3,5-triazinan-1-yl)-4-fluorophenyl)-5-methyl-4,5-dihydroisoxazole-5-carboxylate, 5a, with a Ki value of 4.9 nM, displayed over 2- and 6-fold higher potency than saflufenacil (Ki = 10 nM) and trifludimoxazin (Ki = 31 nM), respectively. Moreover, 5a showed excellent and broad-spectrum weed control against 32 kinds of weeds at 37.5-75 g ai/ha. Rice exhibited relative tolerance to 5a at 150 g ai/ha by postemergence application, indicating that 5a could be a potential herbicide candidate for weed control in paddy fields.


Assuntos
Inibidores Enzimáticos/farmacologia , Herbicidas/química , Herbicidas/farmacologia , Proteínas de Plantas/antagonistas & inibidores , Protoporfirinogênio Oxidase/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/química , Cinética , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Daninhas/química , Plantas Daninhas/efeitos dos fármacos , Plantas Daninhas/enzimologia , Protoporfirinogênio Oxidase/química , Protoporfirinogênio Oxidase/metabolismo , Relação Quantitativa Estrutura-Atividade , Tabaco/química , Tabaco/efeitos dos fármacos , Tabaco/enzimologia , Controle de Plantas Daninhas
2.
J Agric Food Chem ; 67(33): 9254-9264, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31356740

RESUMO

In continuation of our search for potent protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) inhibitors, we designed and synthesized a series of novel herbicidal cycloalka[d]quinazoline-2,4-dione-benzoxazinones. The bioassay results of these synthesized compounds indicated that most of the compounds exhibited very strong Nicotiana tabacum PPO (NtPPO) inhibition activity. More than half of the 37 synthesized compounds displayed over 80% control of all three tested broadleaf weeds at 37.5-150 g ai/ha by postemergent application, and a majority of them showed no phytotoxicity toward at least one kind of crop at 150 g ai/ha. Promisingly, 17i (Ki = 6.7 nM) was 6 and 4 times more potent than flumioxazin (Ki = 46 nM) and trifludimoxazin (Ki = 31 nM), respectively. Moreover, 17i displayed excellent, broad-spectrum herbicidal activity, even at levels as low as 37.5 g ai/ha, and it was determined to be safe for wheat at 150 g ai/ha in postemergent application, indicating the great potential for 17i development as a herbicide for weed control in wheat fields.


Assuntos
Benzoxazinas/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Herbicidas/química , Herbicidas/farmacologia , Proteínas de Plantas/antagonistas & inibidores , Protoporfirinogênio Oxidase/antagonistas & inibidores , Quinazolinas/química , Benzoxazinas/farmacologia , Desenho de Drogas , Cinética , Proteínas de Plantas/química , Plantas Daninhas/efeitos dos fármacos , Plantas Daninhas/enzimologia , Protoporfirinogênio Oxidase/química , Relação Quantitativa Estrutura-Atividade , Quinazolinas/farmacologia , Tabaco/efeitos dos fármacos , Tabaco/enzimologia , Controle de Plantas Daninhas
3.
J Biol Chem ; 293(32): 12394-12404, 2018 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-29925590

RESUMO

Protoporphyrinogen IX oxidase (PPO), the last enzyme that is common to both chlorophyll and heme biosynthesis pathways, catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX. PPO has several isoforms, including the oxygen-dependent HemY and an oxygen-independent enzyme, HemG. However, most cyanobacteria encode HemJ, the least characterized PPO form. We have characterized HemJ from the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) as a bona fide PPO; HemJ down-regulation resulted in accumulation of tetrapyrrole precursors and in the depletion of chlorophyll precursors. The expression of FLAG-tagged Synechocystis 6803 HemJ protein (HemJ.f) and affinity isolation of HemJ.f under native conditions revealed that it binds heme b The most stable HemJ.f form was a dimer, and higher oligomeric forms were also observed. Using both oxygen and artificial electron acceptors, we detected no enzymatic activity with the purified HemJ.f, consistent with the hypothesis that the enzymatic mechanism for HemJ is distinct from those of other PPO isoforms. The heme absorption spectra and distant HemJ homology to several membrane oxidases indicated that the heme in HemJ is redox-active and involved in electron transfer. HemJ was conditionally complemented by another PPO, HemG from Escherichia coli. If grown photoautotrophically, the complemented strain accumulated tripropionic tetrapyrrole harderoporphyrin, suggesting a defect in enzymatic conversion of coproporphyrinogen III to protoporphyrinogen IX, catalyzed by coproporphyrinogen III oxidase (CPO). This observation supports the hypothesis that HemJ is functionally coupled with CPO and that this coupling is disrupted after replacement of HemJ by HemG.


Assuntos
Coproporfirinogênio Oxidase/metabolismo , Heme/metabolismo , Protoporfirinogênio Oxidase/metabolismo , Synechocystis/enzimologia , Tetrapirróis/metabolismo , Coproporfirinogênio Oxidase/química , Heme/química , Modelos Moleculares , Oxirredução , Protoporfirinogênio Oxidase/química , Tetrapirróis/química
4.
Sci Rep ; 7(1): 8860, 2017 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-28821733

RESUMO

Tyrosinases and catechol oxidases belong to the polyphenol oxidase (PPO) enzyme family, which is mainly responsible for the browning of fruits. Three cDNAs encoding PPO pro-enzymes have been cloned from leaves of Malus domestica (apple, MdPPO). The three pro-enzymes MdPPO1-3 were heterologously expressed in E. coli yielding substantial amounts of protein and have been characterized with regard to their optimum of activity resulting from SDS, acidic and proteolytic activation. Significant differences were found in the kinetic characterization of MdPPO1-3 when applying different mono- and diphenolic substrates. All three enzymes have been classified as tyrosinases, where MdPPO1 exhibits the highest activity with tyramine (kcat = 9.5 s-1) while MdPPO2 and MdPPO3 are also clearly active on this monophenolic substrate (kcat = 0.92 s-1 and kcat = 1.0 s-1, respectively). Based on the activity, sequence data and homology modelling it is proposed that the monophenolase and diphenolase activity of PPOs can be manipulated by the appropriate combination of two amino acids, which are located within the active site cleft and were therefore named "activity controllers".


Assuntos
Aminoácidos/química , Catecol Oxidase/química , Catecol Oxidase/metabolismo , Malus/enzimologia , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/metabolismo , Proteínas Recombinantes , Sequência de Aminoácidos , Catecol Oxidase/genética , Expressão Gênica , Malus/genética , Modelos Moleculares , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/isolamento & purificação , Conformação Proteica , Protoporfirinogênio Oxidase/química , Protoporfirinogênio Oxidase/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato
5.
J Agric Food Chem ; 65(28): 5581-5588, 2017 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-28654285

RESUMO

Tuning the binding selectivity through appropriate ways is a primary goal in the design and optimization of a lead toward agrochemical discovery. However, how to achieve rational design of selectivity is still a big challenge. Herein, we developed a novel computational fragment generation and coupling (CFGC) strategy that led to a series of highly potent and bioselective inhibitors targeting protoporphyrinogen IX oxidase. This enzyme plays a vital role in heme and chlorophyll biosynthesis, which has been proven to be associated with many drugs and agrochemicals. However, existing agrochemicals are nonbioselective, resulting in a great threat to nontargeted organisms. To the best of our knowledge, this is the first bioselective inhibitor targeting the tetrapyrrole biosynthesis pathway. In addition, the candidate showed excellent in vivo bioactivity and much better safety toward humans.


Assuntos
Inibidores Enzimáticos/química , Protoporfirinogênio Oxidase/antagonistas & inibidores , Clorofila/metabolismo , Biologia Computacional , Heme/metabolismo , Humanos , Protoporfirinogênio Oxidase/química , Protoporfirinogênio Oxidase/metabolismo , Tabaco/química , Tabaco/enzimologia
6.
J Agric Food Chem ; 65(26): 5278-5286, 2017 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-28616976

RESUMO

To search for new protoporphyrinogen oxidase (PPO, EC 1.3.3.4) inhibitors with improved bioactivity, a series of novel pyrido[2,3-d]pyrimidine-2,4-dione-benzoxazinone hybrids, 9-13, were designed and synthesized. Several compounds with improved tobacco PPO (mtPPO)-inhibiting and promising herbicidal activities were found. Among them, the most potent compound, 3-(7-fluoro-3-oxo-4-(prop-2-yn-1-yl)-3,4-dihydro-2H-benzo[b][1,4] oxazin-6-yl)-1-methylpyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione, 11q, with a Ki value of 0.0074 µM, showed six times more activity than flumioxazin (Ki = 0.046 µM) against mtPPO. Compound 11q displayed a strong and broad spectrum of weed control at 37.5-150 g of active ingredient (ai)/ha by both post- and pre-emergence application, which was comparable to that of flumioxazin. 11q was safe to maize, soybean, peanut, and cotton at 150 g ai/ha, and selective to rice and wheat at 75 g ai/ha by pre-emergence application, indicating potential applicability in these fields.


Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Herbicidas/síntese química , Herbicidas/farmacologia , Proteínas de Plantas/antagonistas & inibidores , Protoporfirinogênio Oxidase/antagonistas & inibidores , Pirimidinas/química , Benzoxazinas/química , Inibidores Enzimáticos/química , Herbicidas/química , Cinética , Proteínas de Plantas/química , Plantas Daninhas/efeitos dos fármacos , Plantas Daninhas/enzimologia , Protoporfirinogênio Oxidase/química , Relação Estrutura-Atividade , Tabaco/enzimologia
7.
Mol Inform ; 35(10): 476-482, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27712043

RESUMO

Protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) catalyzes the oxidation of protoporphyrinogen IX (protogen IX) to protoporphyrin IX (proto IX) in the haem/chlorophyll biosynthetic pathway. Although extensive studies of PPO have already afforded many insights into its biological function and its significance to agriculture and medicine, details of the enzymatic mechanism as well as the nature of the specific amino acids involved in substrate binding still remain largely unknown due to the lack of structural information about protogen IX binding to PPO. In this study, we carried out a detailed and systematic investigation on the binding mode of protogen IX in the Nicotiana tabacum PPO (mtPPO) by performing a computational docking followed by molecular simulations, quantum mechanics calculations, and an integrated analysis. The proposed binding mode was consistent with experimental studies, and several potential key residues that have not been investigated in previous studies, such as Thr70, Arg233, Ser235, Ser474 and Lys477, were identified. In addition, we compared the binding modes of protogen IX in mtPPO and Homo sapiens PPO, and found their differences. Considering the low sequence identity and the differences of biochemical properties among the PPOs from various species, the investigation could provide a valuable basis for the design of PPO inhibitors with high potency and species-selectivity.


Assuntos
Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Protoporfirinogênio Oxidase/química , Protoporfirinas/química , Conformação Molecular , Estrutura Molecular , Protoporfirinogênio Oxidase/metabolismo , Protoporfirinas/metabolismo
8.
J Agric Food Chem ; 64(3): 552-62, 2016 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-26728549

RESUMO

Protoporphyrinogen oxidase (PPO, E.C. 1.3.3.4) is known as a key action target for several structurally diverse herbicides. As a continuation of our research work on the development of new PPO-inhibiting herbicides, a series of novel 3-(2'-halo-5'-substituted-benzothiazol-1'-yl)-1-methyl-6-(trifluoromethyl)pyrimidine-2,4-diones 9 were designed and synthesized. The bioassay results indicated that a number of the newly synthesized compounds exhibited higher inhibition activity against tobacco PPO (mtPPO) than the controls, saflufenacil and sulfentrazone. Compound 9F-5 was identified as the most potent inhibitor with a Ki value of 0.0072 µM against mtPPO, showing about 4.2-fold and 1.4-fold higher potency than sulfentrazone (Ki = 0.03 µM) and saflufenacil (Ki = 0.01 µM), respectively. An additional green house assay demonstrated that compound 9F-6 (Ki = 0.012 µM) displayed the most promising postemergence herbicidal activity with a broad spectrum even at a concentration as low as 37.5 g of active ingredient (ai)/ha. Maize exhibits relative tolerance against compound 9F-6 at the dosage of 150 g ai/ha, but it is susceptible to saflufenacil even at 75 g ai/ha. Thus, compound 9F-6 exhibits the potential to be a new herbicide for weed control in maize fields.


Assuntos
Inibidores Enzimáticos/farmacologia , Herbicidas/farmacologia , Proteínas de Plantas/química , Protoporfirinogênio Oxidase/química , Tabaco/enzimologia , Inibidores Enzimáticos/química , Herbicidas/síntese química , Herbicidas/química , Cinética , Proteínas de Plantas/metabolismo , Protoporfirinogênio Oxidase/metabolismo , Pirimidinonas/química , Pirimidinonas/farmacologia , Relação Quantitativa Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologia , Tabaco/efeitos dos fármacos , Triazóis/química , Triazóis/farmacologia
9.
Genome Biol Evol ; 6(8): 2141-55, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25108393

RESUMO

Tetrapyrroles such as heme and chlorophyll are essential for biological processes, including oxygenation, respiration, and photosynthesis. In the tetrapyrrole biosynthesis pathway, protoporphyrinogen IX oxidase (Protox) catalyzes the formation of protoporphyrin IX, the last common intermediate for the biosynthesis of heme and chlorophyll. Three nonhomologous isofunctional enzymes, HemG, HemJ, and HemY, for Protox have been identified. To reveal the distribution and evolution of the three Protox enzymes, we identified homologs of each along with other heme biosynthetic enzymes by whole-genome clustering across three domains of life. Most organisms possess only one of the three Protox types, with some exceptions. Detailed phylogenetic analysis revealed that HemG is mostly limited to γ-Proteobacteria whereas HemJ may have originated within α-Proteobacteria and transferred to other Proteobacteria and Cyanobacteria. In contrast, HemY is ubiquitous in prokaryotes and is the only Protox in eukaryotes, so this type may be the ancestral Protox. Land plants have a unique HemY homolog that is also shared by Chloroflexus species, in addition to the main HemY homolog originating from Cyanobacteria. Meanwhile, organisms missing any Protox can be classified into two groups; those lacking most heme synthetic genes, which necessarily depend on external heme supply, and those lacking only genes involved in the conversion of uroporphyrinogen III into heme, which would use a precorrin2-dependent alternative pathway. However, hemN encoding coproporphyrinogen IX oxidase was frequently found in organisms lacking Protox enzyme, which suggests a unique role of this gene other than in heme biosynthesis.


Assuntos
Filogenia , Protoporfirinogênio Oxidase/genética , Protoporfirinas/metabolismo , Animais , Bactérias/enzimologia , Bactérias/genética , Bactérias/metabolismo , Vias Biossintéticas , Heme/genética , Heme/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Oxirredução , Plantas/enzimologia , Plantas/genética , Plantas/metabolismo , Protoporfirinogênio Oxidase/química , Protoporfirinogênio Oxidase/metabolismo , Protoporfirinas/genética , Tetrapirróis/genética , Tetrapirróis/metabolismo
10.
J Agric Food Chem ; 62(29): 7209-15, 2014 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-24983412

RESUMO

The potential of protoporphyrinogen oxidase (PPO) to develop resistance against five PPO-inhibiting herbicides has been studied using computational mutation scanning (CMS) protocol, leading to valuable insights into the resistance mechanisms and structure-resistance relationship of the PPO inhibitors. The calculated shifts in the binding free energies caused by the mutations correlated very well with those derived from the corresponding experimental data obtained from site-directed mutagenesis of PPO, leading to valuable insights into the resistance mechanisms of PPO inhibitors. The calculated entropy change was related to the conformational flexibility of the inhibitor, which demonstrated that inhibitors with appropriate conformational flexibility may inhibit both the wild type and mutants simultaneously. The reasonable correlation between the computational and experimental data further validate that CMS protocol is valuable for predicting resistance associated with amino acid mutations on target proteins.


Assuntos
Herbicidas/farmacologia , Protoporfirinogênio Oxidase/antagonistas & inibidores , Estrutura Molecular , Mutagênese Sítio-Dirigida , Protoporfirinogênio Oxidase/química , Protoporfirinogênio Oxidase/genética
11.
J Bacteriol ; 195(18): 4221-30, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23852872

RESUMO

Salicylidene acylhydrazides (SAHs) inhibit the type III secretion system (T3S) of Yersinia and other Gram-negative bacteria. In addition, SAHs restrict the growth and development of Chlamydia species. However, since the inhibition of Chlamydia growth by SAH is suppressed by the addition of excess iron and since SAHs have an iron-chelating capacity, their role as specific T3S inhibitors is unclear. We investigated here whether SAHs exhibit a function on C. trachomatis that goes beyond iron chelation. We found that the iron-saturated SAH INP0341 (IS-INP0341) specifically affects C. trachomatis infectivity with reduced generation of infectious elementary body (EB) progeny. Selection and isolation of spontaneous SAH-resistant mutant strains revealed that mutations in hemG suppressed the reduced infectivity caused by IS-INP0341 treatment. Structural modeling of C. trachomatis HemG predicts that the acquired mutations are located in the active site of the enzyme, suggesting that IS-INP0341 inhibits this domain of HemG and that protoporphyrinogen oxidase (HemG) and heme metabolism are important for C. trachomatis infectivity.


Assuntos
Proteínas de Bactérias/genética , Chlamydia trachomatis/efeitos dos fármacos , Chlamydia trachomatis/genética , Hidrazinas/farmacologia , Mutação , Protoporfirinogênio Oxidase/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Chlamydia trachomatis/enzimologia , Chlamydia trachomatis/patogenicidade , Farmacorresistência Bacteriana , Células HeLa , Heme/metabolismo , Humanos , Ferro/metabolismo , Ferro/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Protoporfirinogênio Oxidase/química , Protoporfirinogênio Oxidase/metabolismo
12.
Bioorg Med Chem ; 21(11): 3245-55, 2013 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-23623257

RESUMO

Protoporphyrinogen oxidase (PPO, E.C. 1.3.3.4) is the action target for several structurally diverse herbicides. A series of novel 4-(difluoromethyl)-1-(6-halo-2-substituted-benzothiazol-5-yl)-3-methyl-1H-1,2,4-triazol-5(4H)-ones 2a-z were designed and synthesized via the ring-closure of two ortho-substituents. The in vitro bioassay results indicated that the 26 newly synthesized compounds exhibited good PPO inhibition effects with K(i) values ranging from 0.06 to 17.79 µM. Compound 2e, ethyl 2-{[5-(4-(difluoromethyl)-3-methyl-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-6-fluorobenzo-thiazol-2-yl]thio}acetate, was the most potent inhibitor with K(i) value of 0.06 µM against mtPPO, comparable to (K(i)=0.03 µM) sulfentrazone. Further green house assays showed that compound 2f (K(i)=0.24 µM, mtPPO), ethyl 2-{[5-(4-(difluoromethyl)-3-methyl-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-6-fluorobenzothiazol-2-yl]thio}propanoate, showed the most promising post-emergence herbicidal activity with broad spectrum even at concentrations as low as 37.5 gai/ha. Soybean exhibited tolerance to compound 2f at the dosages of 150 gai/ha, whereas they are susceptible to sulfentrazone even at 75 gai/ha. Thus, compound 2f might be a potential candidate as a new herbicide for soybean fields.


Assuntos
Herbicidas/síntese química , Proteínas de Plantas/antagonistas & inibidores , Protoporfirinogênio Oxidase/antagonistas & inibidores , Tiazóis/síntese química , Bioensaio , Herbicidas/química , Herbicidas/farmacologia , Simulação de Acoplamento Molecular , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Daninhas/efeitos dos fármacos , Plantas Daninhas/enzimologia , Plantas Daninhas/crescimento & desenvolvimento , Protoporfirinogênio Oxidase/química , Protoporfirinogênio Oxidase/metabolismo , Soja/efeitos dos fármacos , Soja/enzimologia , Soja/crescimento & desenvolvimento , Especificidade da Espécie , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologia , Tiazóis/química , Tiazóis/farmacologia , Triazóis/química , Triazóis/farmacologia
13.
J Biol Chem ; 288(17): 11731-40, 2013 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-23467411

RESUMO

Defects in the human protoporphyrinogen oxidase (hPPO) gene, resulting in ~50% decreased activity of hPPO, is responsible for the dominantly inherited disorder variegate porphyria (VP). To understand the molecular mechanism of VP, we employed the site-directed mutagenesis, biochemical assays, structural biology, and molecular dynamics simulation studies to investigate VP-causing hPPO mutants. We report here the crystal structures of R59Q and R59G mutants in complex with acifluorfen at a resolution of 2.6 and 2.8 Å. The r.m.s.d. of the Cα atoms of the active site structure of R59G and R59Q with respect to the wild-type was 0.20 and 0.15 Å, respectively. However, these highly similar static crystal structures of mutants with the wild-type could not quantitatively explain the observed large differences in their enzymatic activity. To understand how the hPPO mutations affect their catalytic activities, we combined molecular dynamics simulation and statistical analysis to quantitatively understand the molecular mechanism of VP-causing mutants. We have found that the probability of the privileged conformations of hPPO can be correlated very well with the k(cat)/K(m) of PPO (correlation coefficient, R(2) > 0.9), and the catalytic activity of 44 clinically reported VP-causing mutants can be accurately predicted. These results indicated that the VP-causing mutation affect the catalytic activity of hPPO by affecting the ability of hPPO to sample the privileged conformations. The current work, together with our previous crystal structure study on the wild-type hPPO, provided the quantitative structural insight into human variegate porphyria disease.


Assuntos
Flavoproteínas/química , Proteínas Mitocondriais/química , Mutação de Sentido Incorreto , Porfiria Variegada/enzimologia , Protoporfirinogênio Oxidase/química , Substituição de Aminoácidos , Catálise , Cristalografia por Raios X , Flavoproteínas/genética , Humanos , Proteínas Mitocondriais/genética , Mutagênese Sítio-Dirigida , Porfiria Variegada/genética , Estrutura Terciária de Proteína , Protoporfirinogênio Oxidase/genética
14.
Biochim Biophys Acta ; 1823(9): 1617-32, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22575458

RESUMO

The appearance of heme, an organic ring surrounding an iron atom, in evolution forever changed the efficiency with which organisms were able to generate energy, utilize gasses and catalyze numerous reactions. Because of this, heme has become a near ubiquitous compound among living organisms. In this review we have attempted to assess the current state of heme synthesis and trafficking with a goal of identifying crucial missing information, and propose hypotheses related to trafficking that may generate discussion and research. The possibilities of spatially organized supramolecular enzyme complexes and organelle structures that facilitate efficient heme synthesis and subsequent trafficking are discussed and evaluated. Recently identified players in heme transport and trafficking are reviewed and placed in an organismal context. Additionally, older, well established data are reexamined in light of more recent studies on cellular organization and data available from newer model organisms. This article is part of a Special Issue entitled: Cell Biology of Metals.


Assuntos
Heme/biossíntese , Ferro/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Complexos Multienzimáticos/metabolismo , Animais , Transporte Biológico , Ferroquelatase/química , Ferroquelatase/metabolismo , Heme/química , Hemeproteínas/biossíntese , Humanos , Insetos/metabolismo , Proteínas de Membrana Transportadoras/química , Modelos Moleculares , Complexos Multienzimáticos/química , Ligação Proteica , Protoporfirinogênio Oxidase/química , Protoporfirinogênio Oxidase/metabolismo , Leveduras/metabolismo
15.
J Agric Food Chem ; 59(11): 6172-9, 2011 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-21517076

RESUMO

Discovery of protoporphyrinogen oxidase (PPO, EC 1.3.3.4) inhibitors has been one of the hottest research areas in the field of herbicide development for many years. As a continuation of our research work on the development of new PPO-inhibiting herbicides, a series of novel N-(benzothiazol-5-yl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-diones (1a-p) and N-(benzothiazol-5-yl)isoindoline-1,3-diones (2a-h) were designed and synthesized according to the ring-closing strategy of two ortho-substituents. The bioassay results indicated that some newly synthesized compounds exhibited higher PPO inhibition activity than the control of sulfentrazone. Compound 1a, S-(5-(1,3-dioxo-4,5,6,7-tetrahydro-1H-isoindol-2(3H)-yl)-6-fluorobenzothiazol-2-yl) O-methyl carbonothioate, was identified as the most potent inhibitor with k(i) value of 0.08 µM, about 9 times higher than that of sulfentrazone (k(i) = 0.72 µM). Further green house assay showed that compound 1b, methyl 2-((5-(1,3-dioxo-4,5,6,7-tetrahydro-1H-isoindol-2(3H)-yl)-6-fluorobenzothiazol-2-yl)thio)acetate, exhibited herbicidal activity comparable to that of sulfentrazone even at a concentration of 37.5 g ai/ha. In addition, among six tested crops, wheat exhibited high tolerance to compound 1b even at a dosage of 300 g ai/ha. These results indicated that compound 1b might have the potential to be developed as a new herbicide for weed control of wheat field.


Assuntos
Desenho de Drogas , Inibidores Enzimáticos/síntese química , Herbicidas/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Herbicidas/química , Herbicidas/farmacologia , Humanos , Cinética , Plantas Daninhas/efeitos dos fármacos , Protoporfirinogênio Oxidase/antagonistas & inibidores , Protoporfirinogênio Oxidase/química , Protoporfirinogênio Oxidase/genética , Protoporfirinogênio Oxidase/metabolismo , Relação Estrutura-Atividade
16.
J Comput Aided Mol Des ; 25(3): 213-22, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21259066

RESUMO

Protoporphyrinogen oxidase (PPO, EC 1.3.3.4), which has been identified as a significant target for a great family of herbicides with diverse chemical structures, is the last common enzyme responsible for the seventh step in the biosynthetic pathway to heme and chlorophyll. Among the existing PPO inhibitors, diphenyl-ether is the first commercial family of PPO inhibitors and used as agriculture herbicides for decades. Most importantly, diphenyl-ether inhibitors have been found recently to possess the potential in Photodynamic therapy (PDT) to treat cancer. Herein, molecular dynamics simulations, approximate free energy calculations and hydrogen bond energy calculations were integrated together to uncover the structure-activity relationships of this type of PPO inhibitors. The calculated binding free energies are correlated very well with the values derived from the experimental k (i) data. According to the established computational models and the results of approximate free energy calculation, the substitution effects at different position were rationalized from the view of binding free energy. Some outlier (e.g. LS) in traditional QSAR study can also be explained reasonably. In addition, the hydrogen bond energy calculation and interaction analysis results indicated that the carbonyl oxygen on position-9 and the NO(2) group at position-8 are both vital for the electrostatic interaction with Arg98, which made a great contribution to the binding free energy. These insights from computational simulations are not only helpful for understanding the molecular mechanism of PPO-inhibitor interactions, but also beneficial to the future rational design of novel promising PPO inhibitors.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Myxococcus xanthus/enzimologia , Éteres Fenílicos/química , Éteres Fenílicos/farmacologia , Protoporfirinogênio Oxidase/antagonistas & inibidores , Protoporfirinogênio Oxidase/metabolismo , Ligações de Hidrogênio , Simulação de Dinâmica Molecular , Protoporfirinogênio Oxidase/química , Relação Estrutura-Atividade , Termodinâmica
17.
FASEB J ; 25(2): 653-64, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21048046

RESUMO

Human protoporphyrinogen IX oxidase (hPPO), a mitochondrial inner membrane protein, converts protoporphyrinogen IX to protoporphyrin IX in the heme biosynthetic pathway. Mutations in the hPPO gene cause the inherited human disease variegate porphyria (VP). In this study, we report the crystal structure of hPPO in complex with the coenzyme flavin adenine dinucleotide (FAD) and the inhibitor acifluorfen at a resolution of 1.9 Å. The structural and biochemical analyses revealed the molecular details of FAD and acifluorfen binding to hPPO as well as the interactions of the substrate with hPPO. Structural analysis and gel chromatography indicated that hPPO is a monomer rather than a homodimer in vitro. The founder-effect mutation R59W in VP patients is most likely caused by a severe electrostatic hindrance in the hydrophilic binding pocket involving the bulky, hydrophobic indolyl ring of the tryptophan. Forty-seven VP-causing mutations were purified by chromatography and kinetically characterized in vitro. The effect of each mutation was demonstrated in the high-resolution crystal structure.


Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Porfiria Variegada/genética , Protoporfirinogênio Oxidase/metabolismo , Sequência de Aminoácidos , Flavina-Adenina Dinucleotídeo/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Conformação Proteica , Protoporfirinogênio Oxidase/química , Protoporfirinogênio Oxidase/genética
18.
Chimia (Aarau) ; 65(12): 961-9, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22273380

RESUMO

As the last common enzyme in the biosynthetic pathway leading to heme and chlorophyll, protoporphyrinogen oxidase (PPO; EC 1.3.3.4) is an ideal target for herbicide development. Currently, about 30 PPO inhibitors have been developed as agricultural herbicides. PPO inhibitors have displayed environmentally benign, but advantageous characteristics, including low toxicity, low effective concentration, broad herbicidal spectrum (active against both monocotyledon and dicotyledon weeds), quick onset of action, and long lasting effect. Over the last several years, great achievements have been made in revealing the structural biology of PPO. Five PPO crystal structures, four isolated in enzyme-inhibitor complexes and one in the native form, have been determined, including those from Nicotiana tabacum, Myxococcus Xanthus, Bacillus subtilis, and human. Although PPO inhibitors have been developed for over forty years, we continue to uncover exciting future prospects for novel PPO-inhibiting herbicides. In this review, we have summarized the structures of PPOs from plants, human, and bacteria; the interactions between PPOs and inhibitors; the quantitative structure-activity relationships of PPO inhibitors; and the molecular design of new PPO inhibitors.


Assuntos
Inibidores Enzimáticos/farmacologia , Herbicidas/química , Protoporfirinogênio Oxidase/antagonistas & inibidores , Modelos Moleculares , Conformação Proteica , Protoporfirinogênio Oxidase/química , Relação Quantitativa Estrutura-Atividade
19.
Bioorg Med Chem ; 18(22): 7948-56, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-20934343

RESUMO

The characteristics of low application rates, good crop selectivity, low residue and environmental safety exhibited by Protoporphyrinogen oxidase (PPO, EC 1.3.3.4)-inhibiting herbicides have attracted a world-wide research interests. As continuation of our research work on the development of new PPO inhibitors, a series of mono-carbonyl analogues of cyclic imides, N-phenyl pyrrolidin-2-ones and N-phenyl-1H-pyrrol-2-ones, were designed and synthesized based on previously established DFT-QSAR results. The PPO inhibition activities of 29 newly synthesized compounds were tested and a predictive comparative molecular field analysis (CoMFA) model was established with the conventional correlation coefficient r(2)=0.980 and the cross-validated coefficient q(2)=0.518. According to the CoMFA model, the substituent effects on the PPO inhibition activity were explained reasonably. Further greenhouse assay showed that 2-(4-chloro-2-fluoro-5-propoxy-phenyl)-2,3,4,5,6,7-hexahydro-isoindol-1-one (C(6), k(i)=0.095µM) and 2-(5-allyloxy-4-chloro-2-fluorophenyl)-2,3,4,5,6,7-hexahydro-isoindol-1-one (C(7), k(i)=0.12µM) displayed excellent post-emergency herbicidal activity at the concentration of 150g.ai/ha against seven tested weeds. Due to their high PPO inhibition effect and broad spectrum herbicidal activity, these two compounds have the potential for further study on crop selectivity and field trial. These results confirmed once again that only one of the carbonyl groups of cyclic imides is essential to the PPO inhibition activity.


Assuntos
Inibidores Enzimáticos/síntese química , Herbicidas/síntese química , Protoporfirinogênio Oxidase/antagonistas & inibidores , Pirróis/química , Pirrolidinonas/química , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Herbicidas/química , Herbicidas/farmacologia , Conformação Molecular , Protoporfirinogênio Oxidase/química , Pirróis/síntese química , Pirróis/farmacologia , Pirrolidinonas/síntese química , Pirrolidinonas/farmacologia , Relação Quantitativa Estrutura-Atividade
20.
Proc Natl Acad Sci U S A ; 107(23): 10436-41, 2010 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-20484676

RESUMO

Cellular energy generation uses membrane-localized electron transfer chains for ATP synthesis. Formed ATP in turn is consumed for the biosynthesis of cellular building blocks. In contrast, heme cofactor biosynthesis was found driving ATP generation via electron transport after initial ATP consumption. The FMN enzyme protoporphyrinogen IX oxidase (HemG) of Escherichia coli abstracts six electrons from its substrate and transfers them via ubiquinone, cytochrome bo(3) (Cyo) and cytochrome bd (Cyd) oxidase to oxygen. Under anaerobic conditions electrons are transferred via menaquinone, fumarate (Frd) and nitrate reductase (Nar). Cyo, Cyd and Nar contribute to the proton motive force that drives ATP formation. Four electron transport chains from HemG via diverse quinones to Cyo, Cyd, Nar, and Frd were reconstituted in vitro from purified components. Characterization of E. coli mutants deficient in nar, frd, cyo, cyd provided in vivo evidence for a detailed model of heme biosynthesis coupled energy generation.


Assuntos
Escherichia coli/metabolismo , Heme/biossíntese , Biocatálise , Grupo dos Citocromos b/metabolismo , Transporte de Elétrons , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Flavinas/metabolismo , Modelos Moleculares , Mutação , Nitrato Redutase/metabolismo , Estrutura Terciária de Proteína , Protoporfirinogênio Oxidase/química , Protoporfirinogênio Oxidase/metabolismo
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