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. 2014 Oct 17;9(10):2255-62.
doi: 10.1021/cb500485w. Epub 2014 Aug 5.

Transition-state analysis of 2-O-acetyl-ADP-ribose hydrolysis by human macrodomain 1

Brett M Hirsch  1 , Emmanuel S Burgos, Vern L Schramm
Affiliations

Transition-state analysis of 2-O-acetyl-ADP-ribose hydrolysis by human macrodomain 1

Brett M Hirsch et al. ACS Chem Biol. .

Abstract

Macrodomains, including the human macrodomain 1 (MacroD1), are erasers of the post-translational modification of monoadenosinediphospho-ribosylation and hydrolytically deacetylate the sirtuin product O-acetyl-ADP-ribose (OAADPr). OAADPr has been reported to play a role in cell signaling based on oocyte microinjection studies, and macrodomains affect an array of cell processes including transcription and response to DNA damage. Here, we investigate human MacroD1 by transition-state (TS) analysis based on kinetic isotope effects (KIEs) from isotopically labeled OAADPr substrates. Competitive radiolabeled-isotope effects and mass spectrometry were used to obtain KIE data to yield intrinsic KIE values. Intrinsic KIEs were matched to a quantum chemical structure of the TS that includes the active site residues Asp184 and Asn174 and a structural water molecule. Transition-state analysis supports a concerted mechanism with an early TS involving simultaneous nucleophilic water attack and leaving group bond cleavage where the breaking C-O ester bond=1.60 Å and the C-O bond to the attacking water nucleophile=2.30 Å. The MacroD1 TS provides mechanistic understanding of the OAADPr esterase chemistry.

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Figures

Figure 1
Figure 1
MacroD1 catalyzes the hydrolysis of the sirtuin product O-acetyl-ADP-ribose by hydrolysis of the 2-O-ester bond to form ADP-ribose and acetate.
Figure 2
Figure 2
Ground and transition state structures. (A) Optimized reactant state of OAADPr in a water PCM model. (B) In vacuo modeled transition-state based on m062x/6-31g(d,p) DFT calculations to best match the intrinsic KIE values. Carbon, hydrogen, oxygen, and nitrogen atoms, are represented in gray, white, red, and blue, respectively. Fragments of Asn174 (below) and Asp184 (right) were included in the QM computational region.
Scheme 1
Scheme 1. Potential MacroD1 Mechanisms for 2-O-AADPr Hydrolysis
(A) The concerted mechanism for MacroD1 includes groups partially bonded to the reaction center at the TS (red). Nucleophilic participation and ester bond loss are both significant. This TS provides the best match to the intrinsic KIEs. (B) A tetrahedral intermediate mechanism (blue) was considered with TS1 or TS2 as rate-limiting steps. The mechanisms in B were eliminated as the intrinsic KIEs do not match the KIEs calculated for these TS structures.
Figure 3
Figure 3
Electrostatic potential surface map of the MacroD1 transition-state. The Asp184 and Asn174 from MacroD1 are labeled. The map is calculated from Gaussian09 electron density and potential maps. Red indicates a relative electron enrichment, whereas blue represents an electron deficiency relative to the reactant.
Figure 4
Figure 4
C1-methoxy-ADPriboside detection. (A) The red arrow indicates where the C1-methoxy-ADPriboside species would appear as a methanolysis product [M–H]−m/z = 572.09, if the mechanism proceeds through a ribocation ion mechanism. (B) Expected peak height of methoxy-ADPriboside is represented in red based on observed ADPr peak response (m/z = 558.06).
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