Predicting enzyme metalation to identify new therapeutic targets in infectious diseases

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Microbial metalloenzymes defend against host stress responses and synthesize nutrients required for pathogenesis. Metalloenzymes unique to microbial metabolism are therefore attractive targets for therapeutic intervention, but inhibitor discovery requires knowledge of the physiological metal cofactor. Critically, the biologically important metal does not always bind the enzyme of interest most tightly when studied in the test tube, a consequence of the Irving-Williams series of transition metal-ligand stabilities. This apparent paradox is overcome in cells by buffering metal availability to differing levels. We have recently developed the tools to determine these buffered levels and thereby overcome the challenge of identifying the physiological metal cofactor of a variety of metalloproteins. Clostridium difficile, the leading cause of hospital-acquired diarrhoea, undergoes sporulation as an essential part of its transmission cycle. Indeed, spores are the infective agent, responsible for infection as well as recurrency. Global regulation of sporulation is known but the requirement for and supply of nutrients, including metals, during this process is poorly studied. Sporulating pathogens therefore offer unique metalloenzyme therapeutic targets that, when inhibited, would limit the spread of infection due to diminished sporulation. To determine the correct metal cofactors of Clostridium enzymes essential for sporulation, metal availabilities will first be determined using our recently developed approach. Clostridium metalloregulatory proteins will be overexpressed and purified to enable determination of metal and DNA-binding affinities using spectroscopic methods. These affinity values will be used to define physiologically available metal levels within the cell. Then, metal affinities of purified Clostridium enzymes will be determined to identify the physiological metal cofactor, ie, the one that matches calculated availability. A complementary line of investigation will examine the relationship between metal availability and nutrient supply on Clostridium sporulation efficiency by focusing on known biosynthetic pathways containing one or more key metalloenzymes.