Ntn-hydrolases unveiled. Structural investigations into isopencillin N acyltransferase and the quorum-quenching acylase PvdQ

PhD ceremony: Mr. M. Bokhove, 14.45 uur, Academiegebouw, Broerstraat 5, Groningen

Thesis: Ntn-hydrolases unveiled. Structural investigations into isopencillin N acyltransferase and the quorum-quenching acylase PvdQ

Promotor(s): prof. B.W. Dijkstra

Faculty: Mathematics and Natural Sciences

This thesis describes the three-dimensional structures of two enzymes that belong to the Ntn-hydrolase family. Ntn-hydrolases are initially produced as inactive precursors. At the moment an Ntn-hydrolase is correctly folded, it cleaves itself into two chains, and the N-terminus of the newly formed chain will be the catalytic center. The two enzymes studied in this thesis are the quorum quenching acylase PvdQ from the pathogenic Pseudomonas aeruginosa and the isopenicillin acyltransferase (AT) from the penicillin producing Penicillium chrysogenum.

Chapters 2 and 3 describe the crystallization and structure elucidation of PvdQ, which is able to disrupt bacterial communication by breaking down signaling molecules. Many pathogenic bacteria use these signaling molecules for communication purposes to plan their strategy of attack. The structure of PvdQ provides insights how PvdQ recognizes and breaks down specific signaling molecules. Furthermore, the structure of PvdQ gives valuable clues how PvdQ can be altered to recognize and degrade signaling molecules of different pathogenic bacteria.

Chapter 4 describes the crystal structure of AT, which is involved in the biosynthesis of penicillin. AT is of interest for a more environmentally friendly synthesis route for antibiotics. We have solved the precursor structure of AT, which provides insights into the mechanism behind self-activation. Using substrate soaking experiments and computer modeling we were able to propose a mechanism for penicillin biosynthesis. Furthermore, the elucidation of the crystal structure and catalytic mechanism provide insights on how we can alter AT to produce novel antibiotics.