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The origin of the electrostatic perturbation in acetoacetate decarboxylase


Acetoacetate decarboxylase (AADase) has long been cited as the prototypical example of the marked shifts in the pKa values of ionizable groups that can occur in an enzyme active site. In 1966, it was hypothesized that in AADase the origin of the large pKa perturbation (-4.5 log units) observed in the nucleophilic Lys 115 results from the proximity of Lys 116, marking the first proposal of microenvironment effects in enzymology. The electrostatic perturbation hypothesis has been demonstrated in a number of enzymes, but never for the enzyme that inspired its conception, owing to the lack of a three-dimensional structure. Here we present the X-ray crystal structures of AADase and of the enamine adduct with the substrate analogue 2,4-pentanedione. Surprisingly, the shift of the pKa of Lys 115 is not due to the proximity of Lys 116, the side chain of which is oriented away from the active site. Instead, Lys 116 participates in the structural anchoring of Lys 115 in a long, hydrophobic funnel provided by the novel fold of the enzyme. Thus, AADase perturbs the pKa of the nucleophile by means of a desolvation effect by placement of the side chain into the protein core while enforcing the proximity of polar residues, which facilitate decarboxylation through electrostatic and steric effects.

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Figure 1: The AADase tertiary structure depicted as a ribbon diagram.
Figure 2: The positions of Lys 115 and Lys 116 and surrounding environment.
Figure 3: The proposed mechanism of AADase.
Figure 4: The biological dodecamer of AADase built from the crystallographic asymmetric unit.

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Protein Data Bank

Data deposits

The coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 3BH2, 3BGT and 3BH3 corresponding, respectively, to C. acetobutylicum acetoacetate decarboxylase and C. violaceum acetoacetate decarboxylase in the unliganded form and complexed with 2,4-pentanedione.


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We thank A. Murzin for provisional classification of the AADase fold and C. Akey for valuable advice on the execution and interpretation of the electron microscopy work. We also thank H. Robinson and A. Soares for help with data collection. This work was supported by a grant to K.N.A. from the National Science Foundation. Data for this study were measured at Beamlines X12B, X25 and X29A of the National Synchrotron Light Source. Financial support comes principally from the Offices of Biological and Environmental Research (BER) and of Basic Energy Sciences (BES) of the US Department of Energy (DOE), and from the National Center for Research Resources (NCRR) of the National Institutes of Health (NIH). Small-angle X-ray scattering analyses were carried out at the Stanford Synchrotron Radiation Lightsource, funded by DOE, BES. The SSRL Structural Molecular Biology Program is supported by DOE, BER, and by NIH, NCRR. The contents of this work are solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH.

Author Contributions M.-C.H. cloned, expressed, purified, crystallized, collected data and performed crystal structure determination, refinement and model analysis. H.T. designed, executed, analysed and wrote the description of the small-angle X-ray scattering analysis. J.F.M. designed, executed, analysed and wrote the description of the electron microscopy experiments. K.N.A. conceived of and designed the project. M.-C.H. and K.N.A. wrote the manuscript; all authors discussed the results and commented on the manuscript.

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Correspondence to Karen N. Allen.

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Supplementary Information

This file contains Supplementary Figures S1-S8 with Legends, Supplementary Tables S1-S4 and a Supplementary Movie Legend. (PDF 1049 kb)

Supplementary Movie

This movie depicts the X-ray crystallographic structure of acetoacetate decarboxylase in the EM density (see file s1 for full Legend). (MP4 3979 kb)

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Ho, MC., Ménétret, JF., Tsuruta, H. et al. The origin of the electrostatic perturbation in acetoacetate decarboxylase. Nature 459, 393–397 (2009).

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