With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Nicholas Silvaggi from University of Wisconsin-Milwaukee to study how protein structure affects its function. In particular he is studying an enzyme known as MppP which reacts with molecular oxygen to add a hydroxyl group into the amino acid arginine. What is most interesting about MppP is that, while its structure closely resembles that of several other related proteins, its chemistry is very different. By studying MppP the PI is learning how to make educated guesses about the functions of the many uncharacterized proteins uncovered in genome sequencing. In addition, understanding how MppP works is improving understanding of natural products biosynthesis and the processes by which existing enzyme structures are adapted to perform new functions. As part of this project the PI is also developing the PX Lab experience, a program designed to give exceptional students from area high schools an immersive experience in modern structural biology research. The students are working as a team with their teachers, and supervised by the PI and his graduate students, to clone, express, purify, crystallize, and determine the structure of a fluorescent protein. In this way, the research being done in the lab is also training teachers and tomorrow's scientists.

The non-proteinogenic amino acid L-enduracididine (L-End) is a component of a number of bacterially-produced natural products. The pathway for the production of L-End from arginine is thought to involve some unique enzymatic activities, but the reactions catalyzed by the three biosynthetic enzymes, MppP, MppQ and MppR, are unknown. Recent findings show that MppP is a previously unknown class of oxygenase that requires only PLP and molecular oxygen to insert an oxygen atom into an unactivated C-H bond. This is an unprecedented activity for a PLP-dependent enzyme. The objective of this work is to understand how MppP catalyzes this reaction and which structural features account for its unusual activity.Ppre-steady state enzyme kinetics, together with kinetic isotope effects, NMR spectroscopy, and mass spectrometry are being used to probe the catalytic mechanism. Structural features of MppP required for its hydroxylation reaction is being identified by X-ray crystallographic and enzymes kinetics studies of mutant forms of MppP, both alone and in complexes with ligands. The outcome of the research is detailed mechanistic information about how MppP carries out its reaction, which is helping understand how evolution has modified the Type I aminotransferase fold to perform a new catalytic function. These outcomes are expanding knowledge of PLP-dependent enzymes, specifically, of enzyme structure-function relationships, as well as improving the accuracy of protein function predictions.

National Science Foundation (NSF)
Division of Chemistry (CHE)
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Max Funk
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University of Wisconsin Milwaukee
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