Zinc-dependent histone deacetylase 8 (HDAC8) catalyzes the removal of acetyl moieties from histone tails, and is critically involved in regulating chromatin structure and gene expression. The detailed knowledge of its catalytic process is of high importance since it has been established as a most promising target for the development of new anti-tumor drugs. By employing BornOppenheimer ab initio QM/MM molecular dynamics simulations and umbrella sampling, a stateof-the-art approach to simulate enzyme reactions, we have provided further evidences against the originally proposed general acid-base catalytic pair mechanism for Zinc-dependent histone deacetylases. Instead, our results indicated that HDAC8 employs a proton-shuttle catalytic mechanism, in which a neutral His143 first serves as the general base to accept a proton from the zinc-bound water molecule in the initial rate-determining nucleophilic attack step, and then shuttles it to the amide nitrogen atom to facilitate the cleavage of the amide bond. During the deacetylation process, the Zn 2+ ion changes its coordination mode and plays multiple catalytic roles. For the K + ion, which is located about 7 Å from the catalytic Zn 2+ ion and conserved in class I and II HDACs, our simulations indicated that its removal would lead to the different transition state structure and a higher free energy reaction barrier for the rate-determining step. It is found that the existence of this conserved K + ion would enhance the substrate binding, increase the basicity of His143, strengthen the catalytic role of zinc ion and improve the transition state stabilization by the enzyme environment.

It is of significant biological interest and medical importance to develop class- and isoform-selective histone deacetylases (HDAC) modulators. The impact of the linker component on HDAC inhibition specificity was revealed, but has not been understood. Herein with Born-Oppenheimer ab initio QM/MM molecular dynamics simulations, a state-of-the-art approach to simulating metallo-enzymes, we have found that the hydroxamic acid remains to be protonated upon its binding to HDAC8, and thus disapproved the mechanistic hypothesis that the distinct zinc-hydroxamate chelation modes between two HDAC subclasses come from different protonation states of the hydroxamic acid. Instead, our simulations suggested a novel mechanism that the chelation mode of hydroxamate with the zinc ion in HDACs is modulated by water access to the linker binding channel. This new insight into the interplay between the linker binding and the zinc chelation emphasizes the importance and gives guidance regarding the linker design for the development of new class-IIa specific HDAC inhibitors.

AbstracsThe different coordination modes and fast ligand exchange of zinc coordination has been suggested to be one key catalytic feature of the zinc ion which makes it an invaluable metal in biological catalysis. However, partly due to the well known difficulties for zinc to be characterized by spectroscopy methods, evidence for dynamic nature of the catalytic zinc coordination has so far mainly been indirect. In this work, Born-Oppenheimer ab initio QM/MM molecular dynamics simulation has been employed, which allows for a first-principle description of the dynamics of the metal active site while properly including effects of the heterogeneous and fluctuating protein environment. Our simulations have provided direct evidence regarding inherent flexibility of the catalytic zinc coordination shell in Thermolysin (TLN) and Histone Deacetylase 8 (HDAC8). We have observed different coordination modes and fast ligand exchange during the picosecond's timescale. For TLN, the coordination of the carboxylate group of Glu166 to Zinc is found to continuously change between monodentate and bidentate manner dynamically; while for HDAC8, the flexibility mainly comes from the coordination to a non-amino-acid ligand. Such distinct dynamics in the zinc coordination shell between two enzymes suggests that the catalytic role of Zinc in TLN and HDAC8 is likely to be different in spite of the fact that both catalyze the hydrolysis of amide bond. Meanwhile, considering that such Born-Oppenheimer ab initio QM/MM MD simulations are very much desired but are widely considered to be too computationally expensive to be feasible, our current study demonstrates the viability and powerfulness of this state-of-the-art approach in simulating metalloenzymes.Zinc is relatively abundant in biological materials. Approximately 10% of the total human proteome have been identified to bind with zinc in vivo from a bioinformatics investigation1 and they play very crucial roles in all forms of life2 -6 . For mononuclear zinc enzymes, a typical metal coordination environment contains three amino acid side chain ligands (His, Glu, Asp and Cys) and one/two small molecule(s). 3, 7 , 8 The flexibility of zinc coordination, which allows different coordination modes and fast ligand exchange, has been suggested to be one key catalytic feature of the zinc ion which makes it an invaluable metal in biological catalysis.9 However, partly due to the well known difficulties for zinc to be characterized by spectroscopy methods 10,11 , evidence for dynamic nature of the catalytic zinc coordination has so far mainly In order to provide deep insights into the dynamics and flexibility of the zinc catalytic site, which would be essential in characterizing their catalytic mechanisms and rational design of novel inhibitors for zinc enzymes, we have carried out DFT QM/MM Born-Oppenheimer molecular dynamics (BOMD) simulations on TLN and HDAC8. Although semi-empirical QM/MM BOMD simulations of some zinc-dependent enzymes have been carried out 29-32, one main concern is the acc...