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

Global shifts, theoretical shifts: changing geographies of religion

10.1177/0309132510362602Progress in Human Geography346755-77

Picturing Peace: Local and Universal Symbols in Three Cultures

Picturing Peace is an ArtsBridge collaboration in which K-12 students learn to use digital cameras to communicate their feelings and ideas about peace. The photographs of three student cultures were analyzed. Both local and universal symbols of peace were found, such as nature, light, community, environment, peace signs, play, spiritual symbols, diversity, body and innocence

Synthesis of NiS and MnS nanocrystals from the molecular precursors (TMEDA)M(SC{O}C6H5)2 (M = Ni, Mn)

10.1021/cg800536wCrystal Growth and Design91352-35

Privacy and Disclosure of Hiv in interpersonal Relationships: A Sourcebook for Researchers and Practitioners

As the HIV epidemic enters its third decade, it remains one of the most pressing health issues of our time. Many aspects of the disease remain under-researched and inadequate attention has been given to the implications for the relationships and daily lives of those affected by HIV. Disclosing an HIV diagnosis remains a decision process fraught with difficulty and despite encouraging medical advances, an HIV diagnosis creates significant anxiety and distress about one\u27s health, self-identity, and close relationships. This book provides an overarching view of existing research on privacy and disclosure while bringing together two significant areas: self-disclosure as a communication process and the social/relational consequences of HIV/AIDS. The unifying framework is communication privacy management and the focus of this volume is on private voluntary relational disclosure as opposed to forced or public disclosure. Utilizing numerous interviews with HIV patients and their families, the authors examine disclosure in a variety of social contexts, including relationships with intimate partners, families, friends, health workers, and coworkers. Of note are the examinations of predictors of willingness to disclose HIV infection, the message features of disclosure, and the consequences of both disclosure and non-disclosure. [Amazon.com]https://digitalcommons.odu.edu/psychology_books/1006/thumbnail.jp

Saturation mutagenesis of putative catalytic residues of benzoylformate decarboxylase provides a challenge to the accepted mechanism

Benzoylformate decarboxylase from Pseudomonas putida (PpBFDC) is a thiamin diphosphate-dependent enzyme that carries out the nonoxidative decarboxylation of aromatic 2-keto acids. The x-ray structure of PpBFDC suggested that Ser-26, His-70, and His-281 would play important roles in its catalytic mechanism, and the S26A, H70A, and H281A variants all exhibited greatly impaired catalytic activity. Based on stopped-flow studies with the alanine mutants, it was proposed that the histidine residues acted as acid-base catalysts, whereas Ser-26 was involved in substrate binding and played a significant, albeit less well defined, role in catalysis. While developing a saturation mutagenesis protocol to examine residues involved in PpBFDC substrate specificity, we tested the procedure on His-281. To our surprise, we found that His-281, which is thought to be necessary for protonation of the carbanion/enamine intermediate, could be replaced by phenyl alanine with only a 5-fold decrease in kcat. Even more surprising were our subsequent observations (i) that His-70 could be replaced by threonine or leucine with approximately a 30-fold decrease in kcat/Km compared with a 4,000-fold decrease for the H70A variant and (ii) that Ser-26, which forms hydrogen bonds with the substrate carboxylate, could be replaced by threonine, leucine, or methionine without significant loss of activity. These results call into question the assigned roles for Ser-26, His-70, and His-281. Further, they demonstrate the danger in assigning catalytic function based solely on results with alanine mutants and show that saturation mutagenesis is a valuable tool in assessing the role and relative importance of putative catalytic residues