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The BBE-catalysed oxidative carbon-carbon bond formation is a new example of the versatility of the flavin cofactor in biochemical reactions. Our goal is to understand the oxidative cyclization reaction by a biochemical and structural approach. We have developed a new expression system for BBE (using cDNA from Eschscholzia california, gold poppy) in Pichia pastoris, which produces large amounts of the protein (ca. 500 mg from a 10-L culture). The availability of suitable quantities of BBE enabled us to crystallize the protein and to solve the structure in collaboration with Prof. Karl Gruber at the Karl-Franzens University Graz (see below). Based on the three-dimensional structure of BBE, we have performed a site-directed mutagenesis program to investigate the role of amino acids present in the active site of the enzyme. In conjunction with other experiments, this has led to the formulation of a new reaction mechanism for the enzyme (thesis project of Andreas Winkler). Currently, Silvia Wallner investigates alternative covalent modifications in the 8α-position in BBE variants that contain either aspartate or tyrosine instead of a histidine. In collaboration with Prof. Toni Kutchan at the Donald Danforth Plant Science Center in St. Louis, we have identified other 8
plant genes that apparently encode flavin-dependent oxidases. These genes are currently expressed in our laboratory in order to characterize the role of the enzymes in alkaloid biosynthesis (thesis project of Silvia Wallner). Dipeptidylpeptidase III Dipeptidyl-peptidases III (DPPIII; EC 3.4.14.4) are Zn-dependent enzymes with molecular masses of ca. 80-85 kDa that specifically cleave the first two amino acids from the N- terminus of different length peptides. All known DPPIII sequences contain the unique motif HEXXGH, which enabled the recognition of the dipeptidyl-peptidase III family as a distinct evolutionary metallopeptidase family (M49). In mammals, DPPIII is associated with important physiological functions such as pain regulation, and hence the enzyme is a potential drug target. Previously, Sigrid Deller and Nina Jajcanin-Jozic have successfully expressed, purified and characterized the recombinant yeast enzyme, and Pravas Baral in Karl Gruber’s laboratory at the Karl-Franzens-University has elucidated the crystal structure of the yeast protein. This work revealed that yeast DPPIII features a novel protein topology. Structure of human DPPIII in its open form (right) and peptide liganded (closed) form In collaboration with a structural genomics group in Toronto led by Dr. Sirano Dhe-Paganon, Gustavo Arruda has solved the structure of the human enzyme both in its open (right, above) and closed conformation (left, above). The latter structure was obtained by co-crystallization of a tightly binding peptide to an inactive variant of human DPPIII. These two new structures constitute a major breakthrough in our effort to understand the physiological role of the enzyme and pave the way for the development of potentially useful inhibitors of the enzyme (Gustavo Arruda’s thesis project in Prof. Gruber’s laboratory supported by Alexandra Binter). 9
Page 1 and 2: Graz University of Technology Austr
Page 3 and 4: Brief History of the Institute of B
Page 5 and 6: Highlights of 2010 In 2010, Peter M
Page 7: Biochemistry group Group leader: Pe
Page 11 and 12: unusual amino acids, i.e. hydroxypy
Page 13 and 14: words, the hydroxyl group attacks t
Page 15 and 16: 2) Jajcanin-Jozic, N., Deller, S.,
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Page 29 and 30: 2.1. Fluorescent suicide inhibitors
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Page 35 and 36: structural characterization of TAGs
Page 37 and 38: Lectures and Laboratory Courses Akt

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