Using saturation mutagenesis to explore substrate specificity and catalysis in benzoylformate decarboxylase

poster abstractBenzoylformate decarboxylase (BFDC) from Pseudomonas putida and pyruvate decarboxylase (PDC) from Zymomonas mobilis are thiamin diphosphate (ThDP)-dependent enzymes. The two share a common three-dimensional structure and catalyze a similar chemical reaction, i.e., decarboxylation of 2-keto acids. However, they vary significantly in their substrate utilization pattern. In particular, BFDC has extremely limited activity with pyruvate, while PDC has no activity with benzoylformate. Both enzymes also catalyze stereospecific carboligation reactions that are of commercial interest, again with a different range of substrates. In order to identify similarities and differences on a molecular level, and to reveal factors responsible for substrate specificity and enantioselectivity, the X-ray structures BFDC and PDC were compared. Residues identified in this process were subjected to site-directed mutagenesis. The results show that, although it was not possible to simply interchange substrates, it was possible to engineer enzymes that had distinctly different substrate specificities while retaining excellent kinetic activity. However, it also became apparent that a more general approach was needed. Towards this end we developed a screening procedure for BFDC to enable us to use saturation mutagenesis to examine residues involved in substrate specificity. During the development of the methodology it became clear that it was possible to use this approach to explore residues involved in catalysis by BFDC. Here we describe the unexpected results obtained using saturation mutagenesis on putative catalytic residues. In addition we report towards converting BFDC into an efficient pyruvate decarboxylase

Structure-function relationships in the Escherichia coli glycogen synthase

La glucógeno sintetasa (GS) de Escherichia coli es una glicosiltransferasa que transfiere glucosas desde la ADP-glucosa (ADP-Glc) hacia el extremo no reductor del glucógeno, reteniendo la configuración anomérica. En el presente trabajo, se identificó mediante técnicas de modificación química y mutagénesis sitio-dirigida una zona conservada entre las GS bacterianas y almidón sintetasas involucrada en catálisis y unión de sustratos. Posteriormente, se generó un modelo de homología de la GS de E. coli basándose en la estructura de otras tres glicosiltransferasas con un mismo tipo de fold GT-B. La comparación entre las estructuras de las glicosiltransferasas cristalizadas y el modelo de homología llevó a la identificación de un grupo de residuos conservados compartiendo una misma topología. Las mutaciones E377A, D137A, R3OOA,K305A y H161A disminuyeron la actividad específica 10000, 8100, 2600, 1200 y 710 veces, respectivamente. Ninguna de las mutaciones aumentó el S0.5; para el glucógeno, y sólo H161A y R300A mostraron S0.5; aumentados para la ADP-Glc en ll y 8 veces, respectivamente. Estos residuos resultaron esenciales para la catálisis enzimática, validando el modelo que muestra una importante similitud en el sitio activo de GS de E. coli y otras glicosiltransferasas con un fold GT-B cristalizadas hasta la fecha. Asimismo, se identificaron con técnicas de modelado y se estudiaron otros residuos conservados que tienen una participación potencial en la unión de la ADP-Glc y el glucógeno. Esta es la primera vez que se estudia detalladamente el sitio activo de una GS bacteriana.Fil:Yep Rodríguez, Alejandra. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Junin virus-induced astrocytosis is impaired by iNOS inhibition

Because Junin virus (JV) experimental encephalitis of mice and rats is characterized by mild histopathological changes that do not seem to justify per se lethality after intracerebral infection, such a murine model seems adequate to investigate the potential role of inducible nitric oxide synthase (iNOS) as a pathogenic factor. Concomitant with a predominant astrocyte reaction, increased immunoperoxidase expression of iNOS, mitochondrial superoxide dismutase (SODm) and glutathione peroxidase (GPX) was disclosed in brain of mice infected with JV strain #44. When specific inhibition of iNOS was achieved by intraperitoneal administration of amino guanidine (AG), significantly greater mortality was observed in treated animals (70% vs. 40%), together with similar infective titers (∼107 PFU/g) but lower astrocytosis, as shown by glial fibrillary acidic (GFAP) labeling. As regards SODm and GPX immunochemical expression in neurons, no differences were found between mice with or without AG treatment. The present results suggest that the apparent protective role of nitric oxide (NO), when synthesized by iNOS, is unrelated to reduced viral replication but rather to enhanced astrocyte activation behaving as a beneficial cell response to virus-induced CNS damage. © 2003 Wiley-Liss, Inc.Fil: Gomez, Ricardo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; ArgentinaFil: Yep, Alejandra. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; ArgentinaFil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Berria, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; Argentin

An assay for adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase

Adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the conversion of glucose 1-phosphate and adenosine 5′-triphosphate to ADP-glucose and pyrophosphate. We present a radioactive assay of this enzyme with a higher signal/noise ratio. After stopping the reaction that uses [14C]glucose 1-phosphate as a substrate, the ADP-[14C]glucose formed as a product is converted to [14C]glycogen by the addition of glycogen synthase and nonradioactive glycogen as primer. The final product is precipitated and washed, and the radioactivity is measured in a scintillation counter. The [ 14C]glucose 1-phosphate that did not react is easily eliminated during the washes. We have found that this assay produces much lower blanks than previously described radioactive methods based on binding of ADP-[ 14C]glucose to O-(diethylaminoethyl)-cellulose paper. In addition, we tested the kinetic parameters for the effectors of the Escherichia coli ADP-Glc PPase and both assays yielded identical results. The presented method is more suitable for Km or S0.5 determinations of ADP-Glc PPases having high apparent affinity for glucose 1-phosphate. It is possible to use a higher specific radioactivity to increase the sensitivity at lower concentrations of [14C]glucose 1-phosphate without compromising the blanks obtained at higher concentrations.Fil: Yep, Alejandra. Michigan State University; Estados UnidosFil: Bejar, Clarisa M.. Michigan State University; Estados UnidosFil: Ballicora, Miguel A.. Michigan State University; Estados UnidosFil: Dubay, Jennifer R.. Michigan State University; Estados UnidosFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Preiss, Jack. Michigan State University; Estados Unido

The Crystal Structures of the Open and Catalytically Competent Closed Conformation of Escherichia coli Glycogen Synthase*

Escherichia coli glycogen synthase (EcGS, EC 2.4.1.21) is a retaining glycosyltransferase (GT) that transfers glucose from adenosine diphosphate glucose to a glucan chain acceptor with retention of configuration at the anomeric carbon. EcGS belongs to the GT-B structural superfamily. Here we report several EcGS x-ray structures that together shed considerable light on the structure and function of these enzymes. The structure of the wild-type enzyme bound to ADP and glucose revealed a 15.2° overall domain-domain closure and provided for the first time the structure of the catalytically active, closed conformation of a glycogen synthase. The main chain carbonyl group of His-161, Arg-300, and Lys-305 are suggested by the structure to act as critical catalytic residues in the transglycosylation. Glu-377, previously thought to be catalytic is found on the α-face of the glucose and plays an electrostatic role in the active site and as a glucose ring locator. This is also consistent with the structure of the EcGS(E377A)-ADP-HEPPSO complex where the glucose moiety is either absent or disordered in the active site