A general understanding in biochemistry is that the molecular structure of an enzyme defines its function and, thus, two enzymes having the same or very similar structures (homologs) should catalyze the same or similar chemical reactions. With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Nicholas Silvaggi from the University of Wisconsin Milwaukee to determine how homologs of an enzyme from two different organisms produce different products using different chemical steps. The pyridoxal-5'-phosphate-dependent L-arginine deaminase (or MppP) enzyme from the organism Streptomyces wadayamensis (SwMppP) was found by Dr. Silvaggi to react with molecular oxygen to add a hydroxyl group onto the amino acid arginine. The enzyme from Paenibacillus elgii (PeMppP) has a nearly identical structure to SwMppP, but instead creates a new double bond in the middle of the arginine side chain. Both products are ultimately incorporated into secondary metabolites including antibiotics. The lessons learned from these studies help to predict the functions of new proteins uncovered through genomic studies, and to better understand how existing enzyme structures adapt to perform new functions. An outreach program is developed by Dr. Silvaggi to improve the scientific literacy of the public. This program produces short video explanations of scientific questions raised by 3rd and 4th grade students, with the goal of showing how science relates in very intimate ways to their everyday lives.
The non-proteinogenic amino acids L-enduracididine (L-End) and 4,5-dehydroarginine (dhArg) are components of bacterially-produced natural products mannopeptimycin and indolmycin, respectively. Both L-End and dhArg are produced by PLP-dependent oxidases. The enzyme from Streptomyces wadayamensis (SwMppP) uses molecular oxygen to install a hydroxyl group at carbon 4 of the L-Arg substrate side chain, while that from Paenibacillus elgii (PeMppP) creates a double bond between carbons 4 and 5 (PeMppP). SwMppP and PeMppP have nearly identical tertiary structures and share identical active site residues. The only structural differences appear to be at the dimer interface, leading to the hypothesis that differences in the quaternary structure impact the shapes of the active sites and thus the binding mode of the L-Arg substrate. A primary question being addressed is how these two structurally similar enzymes synthesize different products through their different chemical mechanisms. Although much is known (or can be inferred) about the mechanisms of SwMppP and PeMppP, neither mechanism has been fully characterized. In this project, the novel alternative substrate 3,4-dehydro-L-arginine is used to bypass the early stages and thus probe the late stages of the catalytic mechanism of SwMppP though stopped-flow and quench-flow kinetics, NMR spectroscopy, and mass spectrometry experiments. A similar strategy is used to study the mechanism of PeMppP. The outcomes of this research will be detailed information on the reaction mechanisms of these two MppP-like proteins and consequently how evolution has modified the Type I aminotransferase fold to perform new catalytic functions. These outcomes expand our knowledge of PLP-dependent enzymes specifically and of enzyme structure-function relationships in general, and improves the accuracy of protein function predictions.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
