Single-cell organisms are some of the planet’s most creative chemists. Through the power of chemistry, they are able to live nearly everywhere, survive an array of chemical challenges, and produce some of nature’s most interesting and useful natural products. In doing so, they often make use of a broad palette of elements and cofactors. Single-celled organisms also change, sometimes quickly, to cope with changing environments. Projects in our lab deal with one or more of these aspects of microbial biochemistry – newly evolved reactions, useful biosynthetic and biodegradative pathways, and the use and systemic handling of elements beyond the “organic six” (C, H, O, N, P, S) that make up the major part of biopolymers. We use a variety of biochemical and physical methods, here and in conjunction with collaborators around the country. We choose challenging projects that build a variety of skills, preparing students for careers in academia, biotech, pharmaceuticals, agriculture, and environmental science

Evolved reactions

It is well-known that antibiotic resistance evolves and spreads rapidly. By analogy, other kinds of chemical challenge, for example a polluted environment, can provoke a bacterial response in a relatively short amount of time. Recently, organisms have been isolated that are capable of using and thereby detoxifying oxochlorates - highly toxic, man-made, and very environmentally pervasive oxidants as of the last 50 years – as respiratory substrates. These include perchlorate, chlorate, and chlorite, which have had broad use as biocides and bleaching agents as well as chemical oxidants. This evolved metabolism represents a new kind of microbicide resistance. Two enzymes are involved in reductively detoxifying these substrates to harmless Cl- and dioxygen. These enzymes may inspire an elegant natural solution to a man-made problem.

Above: Schematic showing some of the many reactions catalyzed by heme-dependent proteins

The second enzyme in the pathway, chlorite dismutase (Cld), catalyzes the heme-dependent conversion of chlorite to Cl- and dioxygen. This enzyme, believed to have evolved its function relatively recently, is the only enzyme other than photosystem II that is known to efficiently generate dioxygen.

We have been defining the novel structural and chemical features of the enzyme that support this new heme-catalyzed reaction. We are also working toward discovering the role of this ancient and widespread heme enzyme family, which clearly extends beyond oxochlorate chemistry.

Above: Active site structure of the chlorite dismutase from Dechloromonas aromatica, solved in collaboration with Brandon Goblirsch and Prof Carrie Wilmot, University of Minnesota.

Biosynthetic pathways

Structurally diverse molecules known as siderophores are responsible for mobilizing and transporting Fe(III) into many bacteria and fungi. Fusarinine siderophores have obligate roles in both iron import and intracellular storage in Aspergillus fumigatus. Medicinally preventing their biosynthesis is one potential way of selectively targeting Aspergillus infections. New antibiotics are critically needed as resistance to current antibiotics rapidly spreads. There is a particular need for new antimicrobial strategies against eukaryotic Aspergillus, as it remains medicinally intractable and deadly in its invasive forms.

Below: a) Reaction catalyzed by L-Orn monooxygenase; b) ferricrocin (left) and fusarinine (right) siderophores used for mobilization/uptake and storage of Fe(III), respectively.

:

The enzyme initiating fusarinine biosynthesis is L-ornithine monooxygenase, which has homologs in a large number of siderophore-producing bacteria. We are currently kinetically characterizing the enzyme and investigating potential inhibition strategies against it.

Challenging chemistry

Complex biochemical systems depend on the interplay of many elements. Iron, an essential element for almost all living things, is also insoluble and therefore highly unavailable in aerobic, neutral environments. The microbes which serve as entry-points for iron into biogeochemical cycles, as well as the microbes which inhabit us, must overcome this challenge in order to survive. We are interested in the mechanisms that microbes use to overcome the challenges presented by iron acquisition. Our work in this area focuses on the environmental Pseudomonad, Pseudomonas mendocina ymp. We have developed molecular biological, genetic, and physiological tools for use with this organism, and have been applying them toward answering environmentally relevant questions. In particular, we have engineered a siderophore(-) mutant of this strain and have used it to investigate the role of siderophores, alone and in conjunction with other important natural organic compounds, in directly solubilizing minerals as nutritional iron sources. Current work is extending these studies to the nanoscale.

:

Our work in this area focuses on the environmental Pseudomonad, Pseudomonas mendocina ymp. We have developed molecular biological, genetic, and physiological tools for use with this organism, and have been applying them toward answering environmentally relevant questions. In particular, we have engineered a siderophore(-) mutant of this strain and have used it to investigate the role of siderophores, alone and in conjunction with other important natural organic compounds, in directly solubilizing minerals as nutritional iron sources. Current work is extending these studies to the nanoscale.