Chemical Modification of 1-Aminocyclopropane Carboxylic Acid (ACC) Oxidase: Cysteine Mutational Analysis, Characterization, and Bioconjugation with a Nitroxide Spin Label.

1-Aminocyclopropane carboxylic acid oxidase (ACCO) catalyzes the last step of ethylene biosynthesis in plants. Although some sets of structures have been described, there are remaining questions on the active conformation of ACCO and in particular, on the conformation and potential flexibility of the C-terminal part of the enzyme. Several techniques based on the introduction of a probe through chemical modification of amino acid residues have been developed for determining the conformation and dynamics of proteins. Cysteine residues are recognized as convenient targets for selective chemical modification of proteins, thanks to their relatively low abundance in protein sequences and to their well-mastered chemical reactivity. ACCOs have generally 3 or 4 cysteine residues in their sequences. By a combination of approaches including directed mutagenesis, activity screening on cell extracts, biophysical and biochemical characterization of purified enzymes, we evaluated the effect of native cysteine replacement and that of insertion of cysteines on the C-terminal part in tomato ACCO. Moreover, we have chosen to use paramagnetic labels targeting cysteine residues to monitor potential conformational changes by electron paramagnetic resonance (EPR). Given the level of conservation of the cysteines in ACCO from different plants, this work provides an essential basis for the use of cysteine as probe-anchoring residues.

General Base-General Acid Catalysis in Human Histone Deacetylase 8.

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.

Variable active site loop conformations accommodate the binding of macrocyclic largazole analogues to HDAC8.

The macrocyclic depsipeptide Largazole is a potent inhibitor of metal-dependent histone deacetylases (HDACs), some of which are drug targets for cancer chemotherapy. Indeed, Largazole partially resembles Romidepsin (FK228), a macrocyclic depsipeptide already approved for clinical use. Each inhibitor contains a pendant side chain thiol that coordinates to the active site Zn(2+) ion, as observed in the X-ray crystal structure of the HDAC8-Largazole complex [Cole, K. E., Dowling, D. P., Boone, M. A., Phillips, A. J., and Christianson, D. W. (2011) J. Am. Chem. Soc. 133, 12474]. Here, we report the X-ray crystal structures of HDAC8 complexed with three synthetic analogues of Largazole in which the depsipeptide ester is replaced with a rigid amide linkage. In two of these analogues, a six-membered pyridine ring is also substituted (with two different orientations) for the five-membered thiazole ring in the macrocycle skeleton. The side chain thiol group of each analogue coordinates to the active site Zn(2+) ion with nearly ideal geometry, thereby preserving the hallmark structural feature of inhibition by Largazole. Surprisingly, in comparison with the binding of Largazole, these analogues trigger alternative conformational changes in loops L1 and L2 flanking the active site. However, despite these structural differences, inhibitory potency is generally comparable to, or just moderately less than, the inhibitory potency of Largazole. Thus, this study reveals important new structure-affinity relationships for the binding of macrocyclic inhibitors to HDAC8.

Compromised structure and function of HDAC8 mutants identified in Cornelia de Lange Syndrome spectrum disorders.

Cornelia de Lange Syndrome (CdLS) is a multiple congenital anomaly disorder resulting from mutations in genes that encode the core components of the cohesin complex, SMC1A, SMC3, and RAD21, or two of its regulatory proteins, NIPBL and HDAC8. HDAC8 is the human SMC3 lysine deacetylase required for cohesin recycling in the cell cycle. To date, 16 different missense mutations in HDAC8 have recently been identified in children diagnosed with CdLS. To understand the molecular effects of these mutations in causing CdLS and overlapping phenotypes, we have fully characterized the structure and function of five HDAC8 mutants: C153F, A188T, I243N, T311M, and H334R. X-ray crystal structures reveal that each mutation causes local structural changes that compromise catalysis and/or thermostability. For example, the C153F mutation triggers conformational changes that block acetate product release channels, resulting in only 2% residual catalytic activity. In contrast, the H334R mutation causes structural changes in a polypeptide loop distant from the active site and results in 91% residual activity, but the thermostability of this mutant is significantly compromised. Strikingly, the catalytic activity of these mutants can be partially or fully rescued in vitro by the HDAC8 activator N-(phenylcarbamothioyl)benzamide. These results suggest that HDAC8 activators might be useful leads in the search for new therapeutic strategies in managing CdLS.

Reactions of amino acids, peptides, and proteins with oxidized metabolites of tris(p-carboxyltetrathiaaryl)methyl radical EPR probes.

Oxidation of the tris(p-carboxyltetrathiaaryl)methyl (TAM) EPR radical probe, TAMa(•), by rat liver microsomes (RLM) + NADPH, or horseradish peroxidase (HRP) + H2O2, or K2IrCl6, led to an intermediate cation, TAMa(+), which was treated with glutathione (GSH), with formation of an adduct, TAMa-SG(•), resulting from the substitution of a TAMa(•) carboxylate group with the SG group. L-α-Amino acids containing a strong nucleophilic residue (NuH), such as L-cysteine or L-histidine, also reacted with TAMa(+), with formation of radical adducts TAMa-Nu(•) in which a carboxylate group of TAMa(•) was replaced with Nu. Other less nucleophilic L-α-amino acids, such as L-arginine, L-serine, L-threonine, L-tyrosine, or L-aspartate, as well as the tetrapeptide H-(Gly)4-OH, reacted with TAMa(+) via their α-NH2 group, with formation of an iminoquinone methide, IQMa, deriving from an oxidative decarboxylation and amination of TAMa(•). Upon reaction of TAMa(+) with L-proline and L-lysine, N-substituted iminoquinone methide adducts, IQMa-Pro and IQMa-Lys, were formed. Finally, preliminary results showed that oxidation of TAMa(•) in the presence of bovine serum albumin (BSA), led to the covalent binding of TAMa-derived metabolites to BSA. Oxidation of another frequently used TAM probe, TAMb(•) (Oxo63), in the presence of GSH, N-acetyl-cysteine methyl ester, or histidine also led to TAMb-Nu(•) adducts equivalent to the corresponding TAMa-Nu(•) adducts, suggesting that the oxidative metabolism of such TAM(•) probes could lead to protein covalent binding. Moreover, the above data describe an easy access to new TAM radical EPR probes coupled to amino acids, peptides or proteins that could be useful for addressing various biological targets.