Mechanism of the conformational transitions in 6-hydroxymethyl-7,8- dihydropterin pyrophosphokinase as revealed by NMR spectroscopy

Guangyu Li, Krzysztof Felczak, Genbin Shi, Honggao Yan

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), leading to the biosynthesis of folate cofactors. HPPK undergoes dramatic conformational changes during its catalytic cycle, and the conformational changes are essential for enzymatic catalysis. Thus, the enzyme is not only an attractive target for developing antimicrobial agents but also an excellent model system for studying the catalytic mechanism of enzymatic pyrophosphoryl transfer as well as the role of protein dynamics in enzymatic catalysis. In the present study, we report the NMR solution structures of the binary complex HPPK⊙MgAMPCPP and the ternary complex HPPK⊙MgAMPCPP⊙DMHP, where α,β-methyleneadenosine triphosphate (AMPCPP) and 7, 7-dimethyl-6-hydroxypterin (DMHP) are the analogues of the substrates ATP and HP, respectively. The results suggest that the three catalytic loops of the binary complex of HPPK can assume multiple conformations in slow exchanges as evidenced by multiple sets of NMR signals for several residues in loops 2 and 3 and the very weak or missing NH cross-peaks for several residues in loops 1 and 3. However, the ternary complex shows only one set of NMR signals, and the cross-peak intensities are rather uniform, suggesting that the binding of the second substrate shifts the multiple conformations of the binary complex to an apparently single conformation of the ternary complex. The NMR behaviors and conformations of the binary complex HPPK⊙MgAMPCPP are significantly different from those of HPPK in complex with Mgβ,γ-methyleneadenosine triphosphate (MgAMPPCP). It is suggested that the conformational properties of the binary substrate complex HPPK⊙MgATP be represented by those of HPPK⊙MgAMPCPP, because MgAMPCPP is a better MgATP analogue for HPPK with respect to both binding affinity and bound conformation.

Original languageEnglish (US)
Pages (from-to)12573-12581
Number of pages9
JournalBiochemistry
Volume45
Issue number41
DOIs
StatePublished - Oct 17 2006

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