tailieunhanh - Báo cáo khoa học: Mechanism of dihydroneopterin aldolase NMR, equilibrium and transient kinetic studies of the Staphylococcus aureus and Escherichia coli enzymes

Dihydroneopterin aldolase (DHNA) catalyzes both the cleavage of 7,8-dihydro-d-neopterin (DHNP) to form 6-hydroxymethyl-7,8-dihydro-pterin (HP) and glycolaldehyde and the epimerization of DHNP to form 7,8-dihydro-l-monapterin (DHMP). | ễFEBS Journal Mechanism of dihydroneopterin aldolase NMR equilibrium and transient kinetic studies of the Staphylococcus aureus and Escherichia coli enzymes Yi Wang Yue Li Yan Wu and Honggao Yan Department of Biochemistry and Molecular Biology Michigan State University East Lansing MI USA Keywords dihydroneopterin aldolase Escherichia coli folate biosynthesis mechanism Staphylococcus aureus Correspondence H. Yan Department of Biochemistry and Molecular Biology Michigan State University East Lansing MI 48824 USA Fax 1 517 353 9334 Tel 1 517 353 5282 E-mail yanh@ Website http faculty These authors have contributed equally to this work Received 13 January 2007 revised 14 February 2007 accepted 28 February 2007 doi Dihydroneopterin aldolase DHNA catalyzes both the cleavage of dihydro-D-neopterin DHNP to form 6-hydroxymethyl-7 8-dihydro-pterin HP and glycolaldehyde and the epimerization of DHNP to form dihydro-L-monapterin DHMP . Whether the epimerization reaction uses the same reaction intermediate as the aldol reaction or the deprotonation and reprotonation of C2 of DHNP has been investigated by NMR analysis of the reaction products in a D2O solvent. No deuteration of C2 was observed for the newly formed DHMP. This result strongly suggests that the epimerization reaction uses the same reaction intermediate as the aldol reaction. In contrast with an earlier observation the DHNA-catalyzed reaction is reversible which also supports a nonstereospecific retroaldol aldol mechanism for the epimerization reaction. The binding and catalytic properties of DHNAs from both Staphylococcus aureus SaDHNA and Escherichia coli EcDHNA were determined by equilibrium binding and transient kinetic studies. A complete set of kinetic constants for both the aldol and epimerization reactions according to a unified kinetic mechanism was determined for both SaDHNA and EcDHNA. The results show that the two enzymes have .