Toward the structure and function of carbon-phosphorus lyase enzymes

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He, ShuMei
structure and function
Organophosphonates are characterized by a very stable carbon-phosphorus bond. Eshcherichia coli and many other strains of bacteria possess a multi-enzyme system called carbon-phosphorus (C-P) lyase that enables these organisms to cleave the C-P bond of organophosphonates when inorganic phosphate is scarce in the local environment. Genetic studies have demonstrated that C-P lyase is encoded by the fourteen-gene phn operon, phnCDEFGHIJKLMNOP. However, the mechanism for C-P bond cleavage is still unclear. We have expressed, purified, and characterized phnP from this operon. PhnP is a phosphodiesterase which will hydrolyze both bis-(p-nitrophenyl) phosphate and 2’:3’-cyclic nucleotides as substrates. In collaboration with Dr. Zongchao Jia and Katarenya Podzelinska (Queen’s Biochemistry), we have crystallized phnP and solved the crystal structure at a resolution of 1.3 Å. The structure displayed similarity to zinc-dependent metallo-β-lactamase family proteins. However, phnP displays unique structural features with two metal binding sites per monomer: the active site containing potentially two manganese ions, and a ‘structural’ site coordinating one zinc ion. Potential active site residues were identified and corresponding point mutations were generated by site-directed mutagenesis. Studies based on the importance of these residues and the knowledge from our high resolution structure will help elucidate the mechanism of phnP as well as its function in the C-P lyase pathway. Furthermore, we performed a broad range of ligand screening for phnH, another key member from the C-P lyase pathway, by ITC experiments, co-crystallization and high throughput ligand screening. However, all the trials for the identification of the true physiological substrate for phnH proved unsuccessful. Although the mechanism of C-P bond cleavage by C-P lyase still remains unclear, we synthesized a fluorescently labelled organophosphonate (FPn) and utilized it for probing the in vivo degradation of the C-P bond by wild type E. coli and various mutants. Analysis by TLC and mass spectrometry demonstrated the production of the expected alkane product. With this promising fluorescent probe, potential intermediates and substrates can be identified for individual C-P lyase enzymes. Taken together, our studies on the C-P lyase pathway will contribute to elucidate the still unknown mechanism of the cleavage of the stable C-P bond.
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