A Phosphate-binding Histidine of Binuclear Metallophosphodiesterase Enzymes Is a Determinant of 2′,3′-Cyclic Nucleotide Phosphodiesterase Activity

摘要: Binuclear metallophosphoesterases are an enzyme superfamily defined by a shared fold and conserved active site. Although many family members have been characterized biochemically or structurally, the physiological substrates rarely known, features that determine monoesterase versus diesterase activity obscure. In case of dual phosphomonoesterase/diesterase CthPnkp, phosphate-binding histidine was implicated as determinant 2′,3′-cyclic nucleotide phosphodiesterase activity. Here we tested this model comparing catalytic repertoires Mycobacterium tuberculosis Rv0805, which has in its site (His98), Escherichia coli YfcE, cysteine at equivalent position (Cys74). We find Rv0805 previously unappreciated function. Indeed, 150-fold more hydrolyzing 2′,3′-cAMP than 3′,5′-cAMP. Changing His98 to alanine asparagine suppressed without adversely affecting hydrolysis bis-p-nitrophenyl phosphate. Further evidence for defining role derives from our ability convert inactive YfcE protein vigorous specific 2′,3′-cNMP introducing lieu Cys74. YfcE-C74H cleaved P-O2′ bond yield 3′-AMP sole product. on other hand, hydrolyzed either P-O3′ mixture 2′-AMP products, with bias toward 3′-AMP. These reaction outcomes contrast cleaves generate exclusively. It appears enzymic can influence orientation cyclic thereby dictate choice leaving group.