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1.
Biochim Biophys Acta ; 1594(1): 27-39, 2002 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-11825606

RESUMO

The oxidation of IMP to XMP is the rate-limiting step in the de novo synthesis of guanine ribonucleotides. This NAD-dependent reaction is catalyzed by the enzyme inosine monophosphate dehydrogenase (IMPDH). Based upon the recent structural determination of IMPDH complexed to oxidized IMP (XMP*) and the potent uncompetitive inhibitor mycophenolic acid (MPA), we have selected active site residues and prepared mutants of human type II IMPDH. The catalytic parameters of these mutants were determined. Mutations G326A, D364A, and the active site nucleophile C331A all abolish enzyme activity to less than 0.1% of wild type. These residues line the IMP binding pocket and are necessary for correct positioning of the substrate, Asp364 serving to anchor the ribose ring of the nucleotide. In the MPA/NAD binding site, significant loss of activity was seen by mutation of any residue of the triad Arg322, Asn303, Asp274 which form a hydrogen bonding network lining one side of this pocket. From a model of NAD bound to the active site consistent with the mutational data, we propose that these resides are important in binding the ribose ring of the nicotinamide substrate. Additionally, mutations in the pair Thr333, Gln441, which lies close to the xanthine ring, cause a significant drop in the catalytic activity of IMPDH. It is proposed that these residues serve to deliver the catalytic water molecule required for hydrolysis of the cysteine-bound XMP* intermediate formed after oxidation by NAD.


Assuntos
IMP Desidrogenase/genética , Sítios de Ligação , Catálise , Hidrólise , IMP Desidrogenase/química , Inosina Monofosfato/química , Modelos Moleculares , Estrutura Molecular , Mutagênese Sítio-Dirigida , NAD/química , Ribonucleotídeos/química , Xantina
2.
J Med Chem ; 48(25): 7960-9, 2005 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-16335920

RESUMO

Acyl carrier protein synthase (AcpS) catalyzes the transfer of the 4'-phosphopantetheinyl group from the coenzyme A to a serine residue in acyl carrier protein (ACP), thereby activating ACP, an important step in cell wall biosynthesis. The structure-based design of novel anthranilic acid inhibitors of AcpS, a potential antibacterial target, is presented. An initial high-throughput screening lead and numerous analogues were modeled into the available AcpS X-ray structure, opportunities for synthetic modification were identified, and an iterative process of synthetic modification, X-ray complex structure determination with AcpS, biological testing, and further modeling ultimately led to potent inhibitors of the enzyme. Four X-ray complex structures of representative anthranilic acid ligands bound to AcpS are described in detail.


Assuntos
Antibacterianos/síntese química , Modelos Moleculares , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Transferases (Outros Grupos de Fosfato Substituídos)/química , ortoaminobenzoatos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Desenho de Fármacos , Farmacorresistência Bacteriana , Bactérias Gram-Positivas/efeitos dos fármacos , Ligantes , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Quantitativa Estrutura-Atividade , Estereoisomerismo , ortoaminobenzoatos/química , ortoaminobenzoatos/farmacologia
3.
J Antibiot (Tokyo) ; 55(3): 288-95, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12014444

RESUMO

A screening system is described that can detect and confirm inhibitors of the late steps of cell wall biosynthesis. The primary high through-put screen monitors induction of beta-lactamase following exposure to samples, in an Escherichia coli envA- strain that carries the beta-lactamase gene from Citrobacter freundii on a plasmid. Positive samples were detected from compound libraries, from natural products libraries, and from fractions of natural products crude preparations. These samples were then subjected to in vitro assays that could detect the incorporation of soluble cell wall precursor into Lipid I, Lipid II, and polymerized cell wall, using a TLC system that was very accurate and unambiguous in detecting known cell wall inhibitors. One partially purified sample containing a novel antibacterial agent derived from natural products was found to inhibit the formation of Lipid I (50% inhibition at < or = 62.5 ng/ml), whereas another partially purified sample also derived from natural products inhibited transglycosylation into cell wall polymer (50% inhibition at < or = 10 microg/ml). This screening system proved to be especially useful because it was sufficiently sensitive and robust to detect inhibitors among samples of crude preparations or varying states of purity.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Parede Celular/efeitos dos fármacos , Peptidoglicano/biossíntese , Transferases , Antibacterianos/isolamento & purificação , Proteínas de Bactérias/antagonistas & inibidores , Produtos Biológicos/isolamento & purificação , Cromatografia em Camada Fina/métodos , Indução Enzimática , Escherichia coli/química , Escherichia coli/enzimologia , Glicosilação , Metabolismo dos Lipídeos , Testes de Sensibilidade Microbiana , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Transferases (Outros Grupos de Fosfato Substituídos) , beta-Lactamases/biossíntese , beta-Lactamases/genética
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