Research
The Dowling Lab uses structural, biochemical, and biophysical techniques to elucidate how proteins perform their amazing functions within the cell and inform upon their mechanisms. The lab primarily studies enzymes and metalloenzymes that can activate molecules for important chemical transformations. The lab is also interested in nucleic acid systems, exploring DNA-binding proteins that are involved in transcriptional regulation and enzymes that chemical modify DNA nucleobases.
To study these systems, the lab combines X-ray crystallography, light scattering, light spectroscopy, calorimetry, binding studies, and biochemical functional assays. We are most recently beginning to use cryoelectron microscopy to complement these studies.
Flavoprotein monooxygenases
Within the broader family of flavoprotein monooxygenases are the two-component monooxygenases, which require an additional reductase protein to generate reduced flavin for their chemistry. These monooxygenases activate molecular oxygen with reduced flavins to perform a number of biologically important chemical transformations, and we are focused on bacterial monooxygenases involved in the breakdown of organosulfur compounds during sulfur starvation. Our focus is currently on the enzymes responsible for degrading alkanesulfonates. Two of these enzymes fall within the recently identified flavoprotein monooxygenase subfamily that utilizes an N5-(hydro)peroxyflavin intermediate to perform their chemistry.
Diagram: TC FMO Overview
Transcripts for TC FMO Overview diagram
Pyrimidine base hypermodification
The DNA nucleobases define the genetic code, but there are many intricate modifications that affect their access and function. Termed epigenetic modifications, we are excited to explore enzymes from bacteriophage that generate chemical modifications of pyrimidines. We are specifically looking at a modification pathway that begins with incorporation of 5-hydroxymethyldeoxyuridine (5hmdU) within the DNA fiber. This modification pathways echoes our interest in natural product biosynthetic pathways, in that it is a modular pathway in which different enzymes have roles to chemically activate, modify, and tailor pyrimidine hypermodifications, all within the intact DNA fiber.
Diagram: 5hmdU hypermodification within double-stranded DNA:activation and group transfer
Transcript 5hmdU hypermodification within double-stranded DNA activation and group transfer
Potential targets for cancer therapeutics
A few projects within the lab are geared towards understanding the behavior of enzymes or proteins that are either upregulated or mutated within different types of cancers. These systems can serve as prognosis markers and possible sites to develop therapeutics, but we must first understand their mechanism and function at the molecular and chemical level. We are exploring a metallophosphatase involved in central and secondary metabolism that is upregulated in cancer. Additionally, we are exploring the molecular behavior of the capicua transcriptional repressor protein, which binds DNA at a conserved site using HMG box and C1 domains. Phosphorylation of capicua during cellular signaling pathways linked to cell growth and development inactivate capicua, resulting in transcription of target genes, and disruption of capicua’s proper functioning is observed in cancer and metastasis.
Diagram: CIC Overview in Cancer
Enzymes in natural product biosynthesis
The Dowling lab has always been interested in the complexities of natural products. Enzymes within these biosynthetic pathways catalyze amazing transformations, generating diverse chemical scaffolds that have served as inspiration in drug development efforts. The Dowling lab is interested in understanding the structural and functional mechanisms of enzymes in these pathways to inform on their bioengineering, particularly towards compounds that may have antibiotic or anticancer activities. Our work has focused on azoline formation with nonribosomal peptide synthetases in the epothilone and yersiniabactin systems.
Diagram: NRPS Cy Domains
Transcripts for NRPS CY Domains diagram