Histone deacetylases (HDACs) are important enzymes for the transcriptional regulation of gene expression in eukaryotic cells. Recent findings suggest that HDACs could be key targets for chemotherapeutic intervention in malignant diseases. A convenient and sensitive fluorogenic assay for HDAC activity would therefore expedite studies of HDAC in transcriptional regulation and in vitro screening for drug discovery. In this study, novel fluorogenic substrates of HDACs were synthesized with an epsilon-acetylated lysyl moiety and an adjacent MCA moiety at the C terminus of the peptide chain. Upon deacetylation of the acetylated lysyl moiety, molecules became substrates for trypsin, which released highly fluorescent AMC molecules in a subsequent step of the assay. The fluorescence increased in direct proportion to the amount of deacetylated substrate molecules, i.e., HDAC activity. The nonisotopic, homogeneous assay is well suited for high-throughput HDAC inhibitor screening.

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The elucidation of mechanisms of chromatin remodeling, particular transcriptional activation, and repression by histone acetylation and deacetylation has shed light on the role of histone deacetylases (HDAC) as a new kind of therapeutic target for human cancer treatment. HDACs, in general, act as components of large corepressor complexes that prevent the transcription of several tumor suppression genes. In addition, they appear to be also involved in the deacetylation of nonhistone proteins. This paper reviews the most recent insights into the diverse biological roles of HDACs as well as the evolution of this important protein family.

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The full-length gene encoding the histone deacetylase (HDAC)-like amidohydrolase (HDAH) from Bordetella or Alcaligenes (Bordetella/Alcaligenes) strain FB188 (DSM 11172) was cloned using degenerate primer PCR combined with inverse-PCR techniques and ultimately expressed in Escherichia coli. The expressed enzyme was biochemically characterized and found to be similar to the native enzyme for all properties examined. Nucleotide sequence analysis revealed an open reading frame of 1,110 bp which encodes a polypeptide with a theoretical molecular mass of 39 kDa. Interestingly, peptide sequencing disclosed that the N-terminal methionine is lacking in the mature wild-type enzyme, presumably due to the action of methionyl aminopeptidase. Sequence database searches suggest that the new amidohydrolase belongs to the HDAC superfamily, with the closest homologs being found in the subfamily assigned acetylpolyamine amidohydrolases (APAH). The APAH subfamily comprises enzymes or putative enzymes from such diverse microorganisms as Pseudomonas aeruginosa, Archaeoglobus fulgidus, and the actinomycete Mycoplana ramosa (formerly M. bullata). The FB188 HDAH, however, is only moderately active in catalyzing the deacetylation of acetylpolyamines. In fact, FB188 HDAH exhibits significant activity in standard HDAC assays and is inhibited by known HDAC inhibitors such as trichostatin A and suberoylanilide hydroxamic acid (SAHA). Several lines of evidence indicate that the FB188 HDAH is very similar to class 1 and 2 HDACs and contains a Zn 2؉ ion in the active site which contributes significantly to catalytic activity. Initial biotechnological applications demonstrated the extensive substrate spectrum and broad optimum pH range to be excellent criteria for using the new HDAH from Bordetella/ Alcaligenes strain FB188 as a biocatalyst in technical biotransformations, e.g., within the scope of human immunodeficiency virus reverse transcriptase inhibitor synthesis.Class 1 and 2 histone deacetylases (HDACs) (19), acetoin utilization proteins, and acetylpolyamine amidohydrolases (APAH) are members of the same superfamily (17) and may very well descend from a common ancestor (14). Sequence comparison reveals that all three classes of proteins share a number of common motifs. Additionally, they show significant functional similarities such as recognition of acetylated aminoalkyl groups and the removal of the acetyl moiety by cleaving an amide bond. Interestingly, the biological role of APAH is still a matter of speculation, although these enzymes are thought to be involved in degradative pathways of polyamines (20), particularly in the deacetylation of acetylpolyamines (23). However, experimental evidence to support this idea is still rather scarce. To date, only the APAH from Mycoplana ramosa has been described in detail. While some APAH were reported to cleave only acetylputrescine in vitro, M. ramosa APAH was described as less specific in the same experiments (23). Besides having a role in the degradative pathways of polyamines, APAH could, i...