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1.
Biochemistry ; 51(1): 511-20, 2012 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-22148640

RESUMO

The multifunctional proline utilization A (PutA) flavoenzyme from Escherichia coli catalyzes the oxidation of proline to glutamate in two reaction steps using separate proline dehydrogenase (PRODH) and Δ(1)-pyrroline-5-carboxylate (P5C) dehydrogenase domains. Here, the kinetic mechanism of PRODH in PutA is studied by stopped-flow kinetics to determine microscopic rate constants for the proline:ubiquinone oxidoreductase mechanism. Stopped-flow data for proline reduction of the flavin cofactor (reductive half-reaction) and oxidation of reduced flavin by CoQ(1) (oxidative half-reaction) were best-fit by a double exponential from which maximum observable rate constants and apparent equilibrium dissociation constants were determined. Flavin semiquinone was not observed in the reductive or oxidative reactions. Microscopic rate constants for steps in the reductive and oxidative half-reactions were obtained by globally fitting the stopped-flow data to a simulated mechanism that includes a chemical step followed by an isomerization event. A microscopic rate constant of 27.5 s(-1) was determined for proline reduction of the flavin cofactor followed by an isomerization step of 2.2 s(-1). The isomerization step is proposed to report on a previously identified flavin-dependent conformational change [Zhang, W. et al. (2007) Biochemistry 46, 483-491] that is important for PutA functional switching but is not kinetically relevant to the in vitro mechanism. Using CoQ(1), a soluble analogue of ubiquinone, a rate constant of 5.4 s(-1) was obtained for the oxidation of flavin, thus indicating that this oxidative step is rate-limiting for k(cat) during catalytic turnover. Steady-state kinetic constants calculated from the microscopic rate constants agree with the experimental k(cat) and k(cat)/K(m) parameters.


Assuntos
Proteínas de Bactérias/química , Proteínas de Escherichia coli/química , Proteínas de Membrana/química , Prolina Oxidase/química , Prolina/química , Multimerização Proteica , 1-Pirrolina-5-Carboxilato Desidrogenase/química , 1-Pirrolina-5-Carboxilato Desidrogenase/fisiologia , Anaerobiose , Proteínas de Bactérias/fisiologia , Bradyrhizobium/enzimologia , Catálise , Cristalografia por Raios X , Escherichia coli/enzimologia , Proteínas de Escherichia coli/fisiologia , Flavina-Adenina Dinucleotídeo/química , Ácido Glutâmico/química , Homeostase/fisiologia , Isomerismo , Cinética , Proteínas de Membrana/fisiologia , Oxirredução , Prolina Oxidase/fisiologia , Multimerização Proteica/fisiologia , Estrutura Terciária de Proteína
3.
Planta ; 223(2): 213-22, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16079997

RESUMO

A gene fis1 from flax (Linum usitatissimum), which is induced in mesophyll cells at the site of rust (Melampsora lini) infection, is also expressed in vascular tissue, particularly in floral structures of healthy plants. This paper reports that the promoter controlling this expression is contained within 282 bp 5' to the coding region and that fis1 gene induction is specifically by the rust pathogen and not by other fungal pathogens or by wounding. The fis1 gene has 73% homology with an Arabidopsis gene which encodes delta-1-pyrroline-5-carboxylate dehydrogenase (P5CDH) which is a part of the proline degradation pathway. Transgenic flax plants that either over-express fis1 or show reduced fis1 expression due to RNA-mediated gene silencing have an unaltered morphology. However, plants with reduced fis1 expression have markedly increased sensitivity to exogenous proline and show alteration in epidermal cell morphology, callose deposition and the production of hydrogen peroxide during proline-induced death. These lines, which show a biologically significant level of fis1 suppression, have an unaltered reaction to either virulent or avirulent rust infections, as do fis1 over-expression lines. These data indicate that the fis1 gene plays a role in proline metabolism and most likely encodes for a P5CDH enzyme. However, the precise role of fis1 and P5C catabolism in the development of rust disease remains unclear.


Assuntos
1-Pirrolina-5-Carboxilato Desidrogenase/genética , 1-Pirrolina-5-Carboxilato Desidrogenase/fisiologia , Basidiomycota/patogenicidade , Linho/enzimologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Prolina/metabolismo , 1-Pirrolina-5-Carboxilato Desidrogenase/biossíntese , Análise Mutacional de DNA , Linho/genética , Linho/microbiologia , Regulação da Expressão Gênica de Plantas , Genes Reporter , Doenças das Plantas/genética , Proteínas de Plantas/biossíntese , Plantas Geneticamente Modificadas/anatomia & histologia , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/metabolismo , Prolina/toxicidade , Regiões Promotoras Genéticas , Ativação Transcricional
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