...To the Silvaggi group, located in the Department of Chemistry and Biochemistry at the University of Wisconsin-Milwaukee. The primary focus of this laboratory is the relationship between enzyme structure and function. In other words, how can enzymes with no apparent structural similarity catalyze exactly the same chemical reactions? Or, for that matter, how can two seemingly identical enzymes catalyze very different reactions? These structure-function relationships are important for engineering enzymes to perform specific tasks, or when trying to determine the evolutionary relationships between enzymes, for example. We use X-ray crystallography, steady and transient-state enzyme kinetics, site directed mutagenesis, and other biophysical techniques like isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS). Please scroll down to read about current research interests, undergraduate research, and the PX Lab Experience.
The over-arching theme of the research currently underway in the Silvaggi laboratory is understanding the interplay between enzyme structure and function. In particular, we are interested in the enzymology of secondary metabolite biosynthesis, especially nonribosomally-produced peptide antibiotics. The first project in the laboratory focused on the anti-MRSA antibiotic enduracidin and led us to investigate the enzymes that transform L-arginine into the non-proteinogenic amino acid L-enduracididine. We have currently worked out the reactions catalyzed by each of the three enzymes in the pathway, MppP, MppQ, and MppR, and made two very exciting findings. First, the protein MppR is very similar in structure to acetoacetate decarboxylase, but does not have this activity. Rather, MppR catalyzes the cyclization of an oxidized arginine derivative to give the ketone form of L-End. Since we demonstrated that MppQ looks and acts like a typical aminotransferase, and MppP looked from sequence analysis to be an aminotransferase as well, it was not clear where the oxidized arginine derivative would come from. As it turns out, MppP is not an aminotransferase and is instead the first known pyridoxal 5’-phosphate (PLP)-dependent hydroxylase. MppP reacts with L-Arg and dioxygen to generate "4-hydroxy-2-ketoarginine." Other PLP-dependent enzymes are known to catalyze oxidative decarboxylation, but MppP is the first hydroxylase. We are now working to define the catalytic mechanisms of MppR and MppP. Our finding with MppR opened an additional line of inquiry in the lab: exploring the range of reactions catalyzed by enzymes of the acetoacetate decarboxylase-like superfamily (ADCSF). Since most enzymes in the ADCSF are generally annotated as decarboxylases on the basis of sequence identity, very little was known about the reactions performed by the majority of ADCSF enzymes. Our work has shown that the ADCSF contains not only cyclases like MppR, but also α-keto acid aldolases. The aldolase family of the ADCSF contains members biased toward aldol condensation (e.g. secondary metabolite biosynthesis) or hydration of unsaturated bonds and potential retro-aldol cleavage (e.g. aromatic hydrocarbon degradation). This work has greatly expanded the ADCSF field and lays the groundwork for future projects aimed at engineering ADCSF enzymes for use in organic synthesis.
Read more about on-going research projects here.
We take undergraduate research very seriously in this lab. In addition to gaining valuable research experience, undergraduates have made a number of important contributions to our work. So far, every publication coming out of this lab has at least one undergraduate as an author. Students should expect to work an average of 10 hours per week, and to gain experience with every phase of modern structural biology research. This includes cloning/subcloning genes, expression of protein in E. coli, purification by Fast Protein Liquid Chromatography (FPLC), protein crystallization, and X-ray diffraction. We do work with undergraduates at all skill levels: people have to get lab experience somehow. However, it is true that students with at least rudimentary laboratory skills (pipeting, calculating and measuring reagents and assembling solutions, etc) will derive more benefit from the experience. For this reason, preference is given to students who have completed Chem 603 or other significant laboratory experience. Whether you need to complete research credits for graduation, or are interested in pursuing a career in the biomedical sciences, we encourage you to stop by the lab to talk.
Get more information about undergraduate research here.
The PX Lab Experience is a brand new program enabled by a generous research grant from the National Science Foundation. It is based on Dr. Hazel Holden’s Project CRYSTAL, which was a wildly popular NSF-funded program exposing middle school students to hands-on research at UW Madison. The UWM version of the program, PX Lab, is designed to target high school students from Milwaukee. We will pair high school teachers with a motivated student to clone, express, purify, crystallize, and determine the structure of one of four fluorescent proteins. In the spring of 2017, Dr. Silvaggi and his graduate students will work with the teachers in order to train them in the required techniques. We will bring in the first group of students, identified by the teachers, during the fall of 2017. The choice of students will be based on motivation and demonstrated aptitude. The PX Lab Experience will run each fall thereafter. The teachers, Dr. Silvaggi, and graduate students will guide the high school students through the process from cloning to model building and refinement. The data collection portion of the program will take advantage of the remote data collection capabilities at the Life Science Collaborative Access Team (LS-CAT) at the Advanced Photon Source.
Learn more about PX Lab here.