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1.
Sci Rep ; 5: 16641, 2015 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-26573329

RESUMO

S-adenosyl-L-homocysteine hydrolase (SAH hydrolase or SAHH) is a highly conserved enzyme that catalyses the reversible hydrolysis of SAH to L-homocysteine (HCY) and adenosine (ADO). High-resolution crystal structures have been reported for bacterial and plant SAHHs, but not mammalian SAHHs. Here, we report the first high-resolution crystal structure of mammalian SAHH (mouse SAHH) in complex with a reaction product (ADO) and with two reaction intermediate analogues-3'-keto-aristeromycin (3KA) and noraristeromycin (NRN)-at resolutions of 1.55, 1.55, and 1.65 Å. Each of the three structures constitutes a structural snapshot of one of the last three steps of the five-step process of SAH hydrolysis by SAHH. In the NRN complex, a water molecule, which is an essential substrate for ADO formation, is structurally identified for the first time as the candidate donor in a Michael addition by SAHH to the 3'-keto-4',5'-didehydroadenosine reaction intermediate. The presence of the water molecule is consistent with the reaction mechanism proposed by Palmer &Abeles in 1979. These results provide insights into the reaction mechanism of the SAHH enzyme.


Assuntos
Adenosil-Homocisteinase/química , Adenosil-Homocisteinase/metabolismo , Adenosina/análogos & derivados , Adenosina/química , Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Biocatálise , Cristalografia por Raios X , Hidrólise , Camundongos , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Estrutura Terciária de Proteína , S-Adenosil-Homocisteína/metabolismo , Alinhamento de Sequência , Especificidade por Substrato
2.
J Med Chem ; 57(21): 8827-38, 2014 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-25254502

RESUMO

1-Deoxy-d-xylulose 5-phosphate reductoisomerase of Plasmodium falciparum (PfIspC, PfDxr), believed to be the rate-limiting enzyme of the nonmevalonate pathway of isoprenoid biosynthesis (MEP pathway), is a clinically validated antimalarial target. The enzyme is efficiently inhibited by the natural product fosmidomycin. To gain new insights into the structure activity relationships of reverse fosmidomycin analogs, several reverse analogs of fosmidomycin were synthesized and biologically evaluated. The 4-methoxyphenyl substituted derivative 2c showed potent inhibition of PfIspC as well as of P. falciparum growth and was more than one order of magnitude more active than fosmidomycin. The binding modes of three new derivatives in complex with PfIspC, reduced nicotinamide adenine dinucleotide phosphate, and Mg(2+) were determined by X-ray structure analysis. Notably, PfIspC selectively binds the S-enantiomers of the study compounds.


Assuntos
Aldose-Cetose Isomerases/antagonistas & inibidores , Fosfomicina/análogos & derivados , Aldose-Cetose Isomerases/metabolismo , Domínio Catalítico , Cristalização , Fosfomicina/síntese química , Fosfomicina/farmacologia , NADP/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Relação Estrutura-Atividade
3.
Yakugaku Zasshi ; 133(5): 527-37, 2013.
Artigo em Japonês | MEDLINE | ID: mdl-23649394

RESUMO

The human malaria parasite Plasmodium falciparum is responsible for the death of more than a million people each year. The emergence of strains of this malaria parasite resistant to conventional drug therapy has stimulated the search for antimalarial compounds with novel modes of action. Here the structure-function relationship studies for two Plasmodium proteins are presented. One example is the structural studies for S-adenosyl-L-homocysteine hydrolase from Plasmodium falciparum (PfSAHH) and the other example is those for 1-deoxy-D-xylulose reductoisomerase from Plasmodium falciparum (PfDXR). In the former study, the clue for design of species specific PfSAHH inhibitors was obtained by the structural comparison of the active site of PfSAHH with that of human SAHH (HsSAHH). Our study revealed that the inhibitor selectivity depends on the difference of only one amino acid residue in the active site; Cys59 in PfSAHH vs. Thr60 in HsSAHH. In the latter study, the inhibition of PfDXR enzyme by fosmidomycin has proved to be efficient in the treatment of uncomplicated malaria in recent clinical trials conducted in Gabon and Thailand. Our crystal structure analyses of PfDXR/inhibitor complexes revealed the molecular basis of fosmidomycin's action in P. falciparum. We expect that the structure-function relationship studies on Plasmodium proteins are useful for developing the more effective antimalarial compounds.


Assuntos
Adenosil-Homocisteinase/antagonistas & inibidores , Aldose-Cetose Isomerases/antagonistas & inibidores , Antimaláricos/química , Desenho de Fármacos , Inibidores Enzimáticos/química , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Animais , Antimaláricos/uso terapêutico , Domínio Catalítico , Ensaios Clínicos como Assunto , Cristalização , Cristalografia , Cisteína , Fosfomicina/análogos & derivados , Fosfomicina/uso terapêutico , Humanos , Conformação Molecular , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Treonina
4.
Artigo em Inglês | MEDLINE | ID: mdl-21505238

RESUMO

Autotaxin (ATX), which is also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (NPP2 or ENPP2) or phosphodiesterase Iα (PD-Iα), is an extracellular lysophospholipase D which generates lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). ATX stimulates tumour-cell migration, angiogenesis and metastasis and is an attractive target for cancer therapy. For crystallographic studies, the α isoform of human ATX was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 3.0 Šresolution from a monoclinic crystal form belonging to space group C2, with unit-cell parameters a = 311.4, b = 147.9, c = 176.9 Å, ß = 122.6°.


Assuntos
Complexos Multienzimáticos/química , Fosfodiesterase I/química , Pirofosfatases/química , Cristalização , Cristalografia por Raios X , Humanos , Diester Fosfórico Hidrolases
5.
Sci Rep ; 1: 9, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22355528

RESUMO

The human malaria parasite Plasmodium falciparum is responsible for the deaths of more than a million people each year. Fosmidomycin has been proven to be efficient in the treatment of P. falciparum malaria by inhibiting 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), an enzyme of the non-mevalonate pathway, which is absent in humans. However, the structural details of DXR inhibition by fosmidomycin in P. falciparum are unknown. Here, we report the crystal structures of fosmidomycin-bound complete quaternary complexes of PfDXR. Our study revealed that (i) an intrinsic flexibility of the PfDXR molecule accounts for an induced-fit movement to accommodate the bound inhibitor in the active site and (ii) a cis arrangement of the oxygen atoms of the hydroxamate group of the bound inhibitor is essential for tight binding of the inhibitor to the active site metal. We expect the present structures to be useful guides for the design of more effective antimalarial compounds.


Assuntos
Aldose-Cetose Isomerases/química , Simulação por Computador , Modelos Químicos , Modelos Moleculares , Plasmodium falciparum/enzimologia , Sítios de Ligação , Inibidores Enzimáticos/química , Humanos , Plasmodium falciparum/química , Ligação Proteica , Conformação Proteica
6.
Artigo em Inglês | MEDLINE | ID: mdl-20208174

RESUMO

The nonmevalonate pathway of isoprenoid biosynthesis present in Plasmodium falciparum is known to be an effective target for antimalarial drugs. The second enzyme of the nonmevalonate pathway, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), catalyzes the transformation of 1-deoxy-D-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol 4-phosphate (MEP). For crystallographic studies, DXR from the human malaria parasite P. falciparum (PfDXR) was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method in the presence of NADPH. X-ray diffraction data to 1.85 A resolution were collected from a monoclinic crystal form belonging to space group C2 with unit-cell parameters a = 168.89, b = 59.65, c = 86.58 A, beta = 117.8 degrees. Structural analysis by molecular replacement is in progress.


Assuntos
Aldose-Cetose Isomerases/química , Complexos Multienzimáticos/química , Oxirredutases/química , Plasmodium falciparum/enzimologia , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/isolamento & purificação , Cristalização , Cristalografia por Raios X , Expressão Gênica , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/isolamento & purificação , Oxirredutases/genética , Oxirredutases/isolamento & purificação
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