5DC7: Crystal Structure Of D176a-y306f Hdac8 In Complex With A Tetrapeptide Substrate

Citation:
Abstract
Histone deacetylases (HDACs) regulate cellular processes such as differentiation and apoptosis and are targeted by anticancer therapeutics in development and in the clinic. HDAC8 is a metal-dependent class I HDAC and is proposed to use a general acid-base catalytic pair in the mechanism of amide bond hydrolysis. Here, we report site-directed mutagenesis and enzymological measurements to elucidate the catalytic mechanism of HDAC8. Specifically, we focus on the catalytic function of Y306 and the histidine-aspartate dyads H142-D176 and H143-D183. Additionally, we report X-ray crystal structures of four representative HDAC8 mutants: D176N, D176N/Y306F, D176A/Y306F, and H142A/Y306F. These structures provide a useful framework for understanding enzymological measurements. The pH dependence of kcat/KM for wild-type Co(II)-HDAC8 is bell-shaped with two pKa values of 7.4 and 10.0. The upper pKa reflects the ionization of the metal-bound water molecule and shifts to 9.1 in Zn(II)-HDAC8. The H142A mutant has activity 230-fold lower than that of wild-type HDAC8, but the pKa1 value is not altered. Y306F HDAC8 is 150-fold less active than the wild-type enzyme; crystal structures show that Y306 hydrogen bonds with the zinc-bound substrate carbonyl, poised for transition state stabilization. The H143A and H142A/H143A mutants exhibit activity that is >80000-fold lower than that of wild-type HDAC8; the buried D176N and D176A mutants have significant catalytic effects, with more subtle effects caused by D183N and D183A. These enzymological and structural studies strongly suggest that H143 functions as a single general base-general acid catalyst, while H142 remains positively charged and serves as an electrostatic catalyst for transition state stabilization.
PDB ID: 5DC7Download
MMDB ID: 136234
PDB Deposition Date: 2015/8/23
Updated in MMDB: 2017/09
Experimental Method:
x-ray diffraction
Resolution: 2.3  Å
Source Organism:
Similar Structures:
Biological Unit for 5DC7: trimeric; determined by author and by software (PISA)
Molecular Components in 5DC7
Label Count Molecule
Protein (1 molecule)
1
Histone Deacetylase 8(Gene symbol: HDAC8)
Molecule annotation
Nucleotide(1 molecule)
2
Fluor-de-lys Tetrapeptide Assay Substrate
Molecule annotation
Chemicals (8 molecules)
1
2
2
2
3
4
* Click molecule labels to explore molecular sequence information.

Citing MMDB
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