University of Pavia

Department of Genetics and Microbiology, Protein Crystallography Group

Contact details

Leading scientist: Dr. Andrea Mattevi

Address: via Ferrata 1, 27100 Pavia, Italy

Phone: +39 0382985558

Fax: +39 0382528496

Web address: www.unipv.it/biocry

Qualifications and experience

A common theme to most of the research projects is the investigation of medically relevant enzymes with interesting chemical properties, such as complex multifunctional systems and proteins performing unusual catalytic functions. The core of the research activity is represented by X-ray crystallography, employed to study protein three-dimensional structures. This is complemented by other approaches such as site-directed mutagenesis, analysis of enzyme kinetics and computational chemistry. In recent years, we have been studying the structural biochemistry of a few flavin-dependent monooxygenase. In particular, we have elucidated the first structure of a flavin-dependent Baeyer-Villiger monooxygenase. More recently, we have solved several structures of p-hydroxyphenylacetate monooxygenase, a two component enzyme that revealed important aspects in the mechanism of action monooxygenases. A more general problem that we are investigating is the mechanisms underlying the reactivity of flavoenzymes of oxygen. This is still an unresolved problem in that it is not fully understood why certain flavoenzymes can react efficiently with oxygen (oxidases) whereas others do not react almost at all. We are addressing this question by atomic resolution crystallography (including the usage of “Xenon crystallography” to visualize possible oxygen binding cavities) combined with molecular dynamics simulations.

Facilities

The group has the necessary equipment to perform the proposed research. The equipment includes fermentors, cell disruptors, AKTA systems for protein purification, a microcalorimeter for binding assays, spectrophotometers for enzymatic assays, temperature controlled rooms for crystallization, X-ray generator and detector for diffraction data collection, 10 workstations for crystallographic computing and computer graphics, and a robot for protein crystallization with nanoliter volumes. The group has access to MS and other proteomics facilities available at the biotechnology laboratory of Policlinico S. Matteo in Pavia. Our laboratory has routine access to the facilities of Grenoble (ESRF), Villigen (SLS) and Hamburg (EMBL-DESY).

Role in the project

The team in Pavia will mainly focus on the structural enzymology of the flavoprotein monooxygenases involving the analysis of protein-ligand complexes, catalytic intermediates, and protein mutants (workpackage 1). The relevance of these studies will be twofold: to gain basis knowledge into the reaction mechanism(s) underlying flavin-dependent oxygenation reaction and to guide the design of mutant enzymes with desired biocatalytic properties.

Key publications/patents

• Alfieri A, Ferini F, Ruangchan N, Prongjit M, Chaiyen P, Mattevi A (2007) Structure of a two-component monooxygenase. Proc. Natl. Acad. Sci. 104, 1177-1182.
• De Colibus L, Mattevi A (2006) New frontiers in flavoenzyme structure and mechanism. Curr. Opin. Struc. Biol. 16, 722-728.
• Mattevi A (2006) To be or not to be an oxidase: challenging the oxygen reactivity of flavoenzymes. Trends Biochem. Sci. 31, 276-283.
• Malito E, Alfieri A, Fraaije MW, Mattevi A (2004) Crystal Structure of a Baeyer-Villiger Monooxygenase. Proc. Natl. Acad. Sci., 101, 13157-13162.
• Malito E, Coda A, Bilyeu KD, Fraaije MW, Mattevi A. (2004) Structures of Michaelis and product complexes of plant cytokinin dehydrogenase: implications for flavoenzyme catalysis. J. Mol. Biol. 341, 1237-1249.