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Bioorganometallic mechanism of action, and inhibition, of IspH.

ABSTRACT: We have investigated the mechanism of action of Aquifex aeolicus IspH [E-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase], together with its inhibition, using a combination of site-directed mutagenesis (K ( M ),V (max)), EPR and (1)H, (2)H, (13)C, (31)P, and (57)Fe-electron-nuclear double resonance (ENDOR) spectroscopy. On addition of HMBPP to an (unreactive) E126A IspH mutant, a reaction intermediate forms that has a very similar EPR spectrum to those seen previously with the HMBPP "parent" molecules, ethylene and allyl alcohol, bound to a nitrogenase FeMo cofactor. The EPR spectrum is broadened on (57)Fe labeling and there is no evidence for the formation of allyl radicals. When combined with ENDOR spectroscopy, the results indicate formation of an organometallic species with HMBPP, a pi/sigma "metallacycle" or eta (2)-alkenyl complex. The complex is poised to interact with H(+) from E126 (and H124) in reduced wt IspH, resulting in loss of water and formation of an eta (1)-allyl complex. After reduction, this forms an eta (3)-allyl pi-complex (i.e. containing an allyl anion) that on protonation (at C2 or C4) results in product formation. We find that alkyne diphosphates (such as propargyl diphosphate) are potent IspH inhibitors and likewise form metallacycle complexes, as evidenced by (1)H, (2)H, and (13)C ENDOR, where hyperfine couplings of approximately 6 MHz for (13)C and 10 MHz for (1)H, are observed. Overall, the results are of broad general interest because they provide new insights into IspH catalysis and inhibition, involving organometallic species, and may be applicable to other Fe(4)S(4)-containing proteins, such as IspG.

SUBMITTER: Wang W

PROVIDER: S-EPMC2842026 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Bioorganometallic mechanism of action, and inhibition, of IspH.

Wang Weixue W Wang Ke K Liu Yi-Liang YL No Joo-Hwan JH Li Jikun J Nilges Mark J MJ Oldfield Eric E

Proceedings of the National Academy of Sciences of the United States of America 20100219 10

We have investigated the mechanism of action of Aquifex aeolicus IspH [E-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase], together with its inhibition, using a combination of site-directed mutagenesis (K ( M ),V (max)), EPR and (1)H, (2)H, (13)C, (31)P, and (57)Fe-electron-nuclear double resonance (ENDOR) spectroscopy. On addition of HMBPP to an (unreactive) E126A IspH mutant, a reaction intermediate forms that has a very similar EPR spectrum to those seen previously with the HMBPP ...[more]

PMID: 20173096

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Project description:RationaleThe class Ic antiarrhythmic drug flecainide prevents ventricular tachyarrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by hyperactive RyR2 (cardiac ryanodine receptor) mediated calcium (Ca) release. Although flecainide inhibits single RyR2 channels in vitro, reports have claimed that RyR2 inhibition by flecainide is not relevant for its mechanism of antiarrhythmic action and concluded that sodium channel block alone is responsible for flecainide's efficacy in CPVT.ObjectiveTo determine whether RyR2 block independently contributes to flecainide's efficacy for suppressing spontaneous sarcoplasmic reticulum Ca release and for preventing ventricular tachycardia in vivo.Methods and resultsWe synthesized N-methylated flecainide analogues (QX-flecainide and N-methyl flecainide) and showed that N-methylation reduces flecainide's inhibitory potency on RyR2 channels incorporated into artificial lipid bilayers. N-methylation did not alter flecainide's inhibitory activity on human cardiac sodium channels expressed in HEK293T cells. Antiarrhythmic efficacy was tested utilizing a Casq2 (cardiac calsequestrin) knockout (Casq2-/-) CPVT mouse model. In membrane-permeabilized Casq2-/- cardiomyocytes-lacking intact sarcolemma and devoid of sodium channel contribution-flecainide, but not its analogues, suppressed RyR2-mediated Ca release at clinically relevant concentrations. In voltage-clamped, intact Casq2-/- cardiomyocytes pretreated with tetrodotoxin to inhibit sodium channels and isolate the effect of flecainide on RyR2, flecainide significantly reduced the frequency of spontaneous sarcoplasmic reticulum Ca release, while QX-flecainide and N-methyl flecainide did not. In vivo, flecainide effectively suppressed catecholamine-induced ventricular tachyarrhythmias in Casq2-/- mice, whereas N-methyl flecainide had no significant effect on arrhythmia burden, despite comparable sodium channel block.ConclusionsFlecainide remains an effective inhibitor of RyR2-mediated arrhythmogenic Ca release even when cardiac sodium channels are blocked. In mice with CPVT, sodium channel block alone did not prevent ventricular tachycardia. Hence, RyR2 channel inhibition likely constitutes the principal mechanism of antiarrhythmic action of flecainide in CPVT.

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Bioorganometallic mechanism of action, and inhibition, of IspH.

Publications

Bioorganometallic mechanism of action, and inhibition, of IspH.

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