Kevin Pojasek
Massachusetts Institute of Technology
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Featured researches published by Kevin Pojasek.
Biochemical Journal | 2005
Vikas Prabhakar; Ishan Capila; Carlos J. Bosques; Kevin Pojasek; Ram Sasisekharan
GalAGs (galactosaminoglycans) are one subset of the GAG (glycosaminoglycan) family of chemically heterogeneous polysaccharides that are involved in a wide range of biological processes. These complex biomacromolecules are believed to be responsible for the inhibition of nerve regeneration following injury to the central nervous system. The enzymic degradation of GAG chains in damaged nervous tissue by cABC I (chondroitinase ABC I), a broad-specificity lyase that degrades GalAGs, promotes neural recovery. In the present paper, we report the subcloning of cABC I from Proteus vulgaris, and discuss a simple methodology for the recombinant expression and purification of this enzyme. The originally expressed cABC I clone resulted in an enzyme with negligible activity against a variety of GalAG substrates. Sequencing of the cABC I clone revealed four point mutations at issue with the electron-density data of the cABC I crystal structure. Site-directed mutagenesis produced a clone with restored GalAG-degrading function. We have characterized this enzyme biochemically, including an analysis of its substrate specificity. By coupling structural inspections of cABC I and an evaluation of sequence homology against other GAG-degrading lyases, a set of amino acids was chosen for further study. Mutagenesis studies of these residues resulted in the first experimental evidence of cABC Is active site. This work will facilitate the structure-function characterization of biomedically relevant GalAGs and further the development of therapeutics for nerve regeneration.
Biochemical Journal | 2005
Vikas Prabhakar; Rahul Raman; Ishan Capila; Carlos J. Bosques; Kevin Pojasek; Ram Sasisekharan
cABC I (chondroitinase ABC I) from Proteus vulgaris is a GalAG (galactosaminoglycan) depolymerizing lyase that cleaves its substrates at the glycosidic bond via beta-elimination. cABC I cleaves a particularly broad range of GalAG substrates, including CS (chondroitin sulphate), DS (dermatan sulphate) and hyaluronic acid. We recently cloned and recombinantly expressed cABC I in Escherichia coli, and completed a preliminary biochemical characterization of the enzyme. In the present study, we have coupled site-directed mutagenesis of the recombinant cABC I with a structural model of the enzyme-substrate complex in order to investigate in detail the roles of active site amino acids in the catalytic action of the enzyme. The putative catalytic residues His-501, Tyr-508, Arg-560 and Glu-653 were probed systematically via mutagenesis. Assessment of these mutants in kinetic and end-point assays provided direct evidence on the catalytic roles of these active-site residues. The crystal structure of the native enzyme provided a framework for molecular docking of representative CS and DS substrates. This enabled us to construct recombinant enzyme-substrate structural complexes. These studies together provided structural insights into the effects of the mutations on the catalytic mechanism of cABC I and the differences in its processing of CS and DS substrates. All His-501 mutants were essentially inactive and thereby implicating this amino acid to play the critical role of proton abstraction during catalysis. The kinetic data for Glu-653 mutants indicated that it is involved in a hydrogen bonding network in the active site. The proximity of Tyr-508 to the glycosidic oxygen of the substrate at the site of cleavage suggested its potential role in protonating the leaving group. Arg-560 was proximal to the uronic acid C-5 proton, suggesting its possible role in the stabilization of the carbanion intermediate formed during catalysis.
Journal of Biological Chemistry | 2003
Rahul Raman; James R. Myette; Zachary Shriver; Kevin Pojasek; Ganesh Venkataraman; Ram Sasisekharan
In the previous paper (Myette, J. R., Shriver, Z., Claycamp, C., McLean, M. W., Venkataraman, G., and Sasisekharan, R. (2003) J. Biol. Chem. 278, 12157–12166), we described the molecular cloning, recombinant expression, and preliminary biochemical characterization of the heparin/heparan sulfate 2-O-sulfatase fromFlavobacterium heparinum. In this paper, we extend our structure-function investigation of the 2-O-sulfatase. First, we have constructed a homology-based structural model of the enzyme active site, using as a framework the available crystallographic data for three highly related arylsulfatases. In this model, we have identified important structural parameters within the enzyme active site relevant to enzyme function, especially as they relate to its substrate specificity. By docking various disaccharide substrates, we identified potential structural determinants present within these substrates that would complement this unique active site architecture. These determinants included the position and number of sulfates present on the glucosamine, oligosaccharide chain length, the presence of a Δ4,5-unsaturated double bond, and the exolytic versusendolytic potential of the enzyme. The predictions made from our model provided a structural basis of substrate specificity originally interpreted from the biochemical and kinetic data. Our modeling approach was further complemented experimentally using peptide mapping in tandem with mass spectrometry and site-directed mutagenesis to physically demonstrate the presence of a covalently modified cysteine (formylglycine) within the active site. This combinatorial approach of structure modeling and biochemical studies provides insight into the molecular basis of enzyme function.
Journal of Biological Chemistry | 2004
Gurvan Michel; Kevin Pojasek; Yunge Li; Traian Sulea; Robert J. Linhardt; Rahul Raman; Vikas Prabhakar; Ram Sasisekharan; Miroslaw Cygler
Biochemical and Biophysical Research Communications | 2001
Kevin Pojasek; Zachary Shriver; Patrick Kiley; Ganesh Venkataraman; Ram Sasisekharan
Archive | 2001
Dongfang Liu; Kevin Pojasek; Zachary Shriver; Kristine Holley; Yosuf El-Shabrawi; Ganesh Venkataraman; Ram Sasisekharan
Archive | 2003
Kevin Pojasek; Rahul Raman; Ram Sasisekharan
Archive | 2005
Vikas Prabhakar; Ishan Capila; Rahul Raman; Carlos J. Bosques; Kevin Pojasek; Ram Sasisekharan
Biochemistry | 2000
Kevin Pojasek; Zachary Shriver; Yini Hu; Ram Sasisekharan
Journal of Biological Chemistry | 2002
Kevin Pojasek; Rahul Raman; Patrick Kiley; Ganesh Venkataraman; Ram Sasisekharan