Research Overview
The Liptak group elucidates the mechanisms of bioinorganic enzymes to aid the development of novel antibiotics and catalysts for biomedical and environmental applications. Currently, we are particularly focused on enzymes involved in metal tetrapyrrole biosynthesis and degradation. Several pathogenic bacteria employ heme iron acquisition pathways to procure iron duing infection, and we are examining the mechanisms of the heme degrading enzymes from this pathway to aid antibiotic development. We are also investigating the mechanisms of class II chelatases, which catalyze metal insertion during metal tetrapyrrole biosynthesis, with the aim of designing synthetic enzymes for the biosynthesis of non-natural metal tetrapyrroles for alternative energy applications.

To achieve these goals, we employ biochemical, spectroscopic, and computational approaches. Biological samples are prepared via recombinant protein expression, and key reactive intermediates are isolated using oxygen-free techniques. These intermediates, and their analogues, are characterized using a variety of spectroscopies, including: magentic circular dichroism (MCD), nuclear magnetic resonance (NMR), and electron paramagnetic resonance (EPR). To aid interpretation of our spectroscopic data, we model these species using quantum mechanical calculations.

Metal Tetrapyrrole Degradation
The longest-running project in the Liptak group is our study of non-canonical heme oxygenases from Staphylococcus aureus and Mycobacterium tuberculosis. Canonical heme oxygenases degrade heme to biliverdin products whereas non-canonical enzymes from S. aureus and M. tuberculosis degrade heme to non-biliverdin products. Due to their critical role in heme iron acquisition during infection, S. aureus IsdG and M. tuberculosis MhuD are promising antibiotic targets.

We have developed fluorescence- and UV/Vis absorption-based assays to determine that IsdG and MhuD bind a single heme substrate with nanomolar affinity.

We have deduced the structural and functional role of a conserved Asn residue in IsdG. This second-sphere residue properly orients a (hydro)peroxo moiety for regioselective oxygenation of porphyrin and induces partial electron transfer from porphyrin to iron.

Most recently, we have elucidated that electronic and functional implications of an unusual "ruffling" deformation of the heme substrate induced by second-sphere interactions in the MhuD active site. When bound to MhuD, heme has access to two distinct substrate conformations with distinct electronic structures and both conformations are required for conversion of heme to mycobilin!

Metal Tetrapyrrole Biosynthesis
A newer project in the Liptak group is concerned with transition metal insertion during metal tetrapyrrole biosynthesis. Heme, vitamin B12, and cofactor F430 have different transition metal centers (Fe, Co, and Ni, respectively) that are inserted by a structurally-related family of enzymes known as class II chelatases. We are working to elucidate the origins of metal specificity by these enzymes with the long-term goal of engineering synthetic chelatases for biosynthesis of non-natural metal tetrapyrroles..

To date, we have discovered that CfbA inserts a labile Ni(II) into a "ruffled" tetrapyrrole during cofactor F430 biosynthesis.

Student Outcomes
Researchers in the Liptak group learn a variety of biochemical, spectroscopic, and computational skills in addition to management of a research project from the first experiments to journal publication. As a result, graduate student alumni have secured jobs in both academia and industry. The majority of undergraduate alumni have moved on to graduate programs in chemistry.