The control of enzymes by use of an external stimulus such as light enables the temporal and spatial regulation of defined chemical reactions in a highly precise manner. In this work we investigated and characterized the reversible photocontrol of a bacterial histone deacetylase-like amidohydrolase (HDAH) from Bordetella/Alcaligenes strain FB188, which holds great potential to control deacetylation reactions of a broad spectrum of substrates in biotechnological and biomedical applications. Several HDAH variants with a single surface accessible cysteine close to the active site were developed and covalently modified by a monofunctional azobenzene-based photoswitch [4-phenylazomaleinanil (4-PAM)]. The enzymatic activity of three HDAH variants (M30C, S20C and M150C) were shown to be controlled by light. The thermal cis-to-trans relaxation of azobenzene conjugated to HDAH was up to 50-fold retarded compared to unbound 4-PAM allowing light pulse switching rather than continuing irradiation to maintain the thermodynamically less stable cis-state of covalently attached 4-PAM.

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The photocontrol of protein function like enzyme activity has been the subject of many investigations to enable reversible and spatiotemporally defined cascading biochemical reactions without the need for separation in miniaturized and parallelized assay setups for academic and industrial applications. A photoswitchable amidohydrolase variant from Bordetella/Alcaligenes with the longest reported half-life (approximately 30 h) for the cis-state of the attached azobenzene group was chosen as a model system to dissect the underlying mechanism and molecular interactions that caused the enormous deceleration of the thermal cis-to-trans relaxation of the azobenzene photoswitch. A systematic site-directed mutagenesis study on the basis of molecular dynamics simulation data was employed to investigate enzyme and thermal cis-to-trans relaxation kinetics in dependence on selected amino acid substitution, which revealed a prominent histidine and a hydrophobic cluster as molecular determinants for the stabilization of the cis-isomer of the attached azobenzene moiety on the protein surface. The nature of the involved interactions consists of polar, hydrophobic, and possibly aromatic Π-Π contributions. The elucidated principles behind the stabilization of the cis-state of azobenzene derivatives on a protein surface can be exploited to design improved biologically inspired photoswitches. Moreover, the findings open the door to highly long-lived cis-states of azobenzene groups yielding improved bistable photoswitches that can be controlled by single light-pulses rather than continuous irradiation with UV light that causes potential photodamage to the employed biomolecules.

IntroductIon The family of histone deacetylases (hdacs) has emerged as an important novel class of targets for pharmaceutical intervention on different types of cancer.1 there are 11 zinc-dependent hdacs subdivided in classes i, iia, iib, and iV and 7 hdacs that depend on nad + , the so-called sirtuins. hdacs and their antagonistic opponents, histone acetyl transferases (hats), regulate a variety of genes and cellular functions through balancing the acetylation status of histones and many nonhistone proteins.2 recently, two inhibitors of hdacs (hdacis), vorinostat (ZolinZa; merck, Whitehouse station, nJ) and romidepsin (istodax; celgene, summit, nJ), have been approved for the treatment of cutaneous t-cell lymphoma by the Us food and drug administration. the therapeutic interest in hdacis is also reflected by more than 87 open clinical trials.3 although cancer will remain the most important therapeutic area of hdacis for years, there is an exceeding body of evidence that hdacs play a pivotal role in immunological networks, inflammation, 4 neurological disorders, 5 memory, 6 and muscle/ heart diseases. 7 in addition, hdac orthologues in parasitic protozoa may serve as malaria and leishmania targets.8 the bacterial histone deacetylase homologue hdah from Bordetella has been discovered by hildmann et al.9,10 and extensively investigated by them and our group.11,12 hdah is structurally and functionally closely related to hdac6. the enzyme shares 35% identity with the second deacetylase domain of hdac6, and the crystal structure of hdah contains a deacetylase domain very similar to other hdac isoforms with resolved structures. 13 moreover, hdah and hdac6 display similar substrate and inhibitor selectivities.10 consequently, hdah has been used as a proven model system for hdac6 and hdacs in general. 12,14 the outstanding potential of hdacis with defined isoform selectivity profiles as drugs against a plurality of diseases vindicates an increased effort in the development of high-throughput capable assays and screening campaigns. histone deacetylases (hdacs) are important epigenetic factors regulating a variety of vital cellular functions such as cell cycle progression, differentiation, cell migration, and apoptosis. consequently, hdacs have emerged as promising targets for cancer therapy. the drugability of hdacs has been shown by the discovery of several structural classes of inhibitors (hdacis), particularly by the recent approval of two hdacis, vorinostat (ZolinZa) and romidepsin (istodax), for the treatment of cutaneous t-cell lymphoma by the Us food and drug administration. the outstanding potential of hdacis, with a defined isoform selectivity profile as drugs against a plurality of diseases, vindicates increased effort in developing highthroughput capable assays for screening campaigns. in this study, a dual-competition assay exploiting changes in fluorescence anisotropy and lifetime was used to screen the loPac (sigma-aldrich, st louis, mo) library against the bacterial histone deacetylase homologue hdah from...