A New Amidohydrolase from Bordetella or Alcaligenes Strain FB188 with Similarities to Histone Deacetylases

Abstract

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...

Human HDAC7 Harbors a Class IIa Histone Deacetylase-specific Zinc Binding Motif and Cryptic Deacetylase Activity

“…This might be due to the general difficulty in defining catalytic properties of HDAC7 and other class IIa HDACs, since their biological substrates are unknown, and it has been shown that HDACs show different catalytic efficiencies with different substrate peptides (38,40,41,44). On the other hand, the fact that the side chain of His-843 points away from the cdHDAC7 active site could also explain the lower activity of cdHDAC7 since in this position His-843 is unlikely to contribute to the orientation of the substrate acetyl oxygen and the stabilization of the tetrahedral oxyanion intermediate during catalysis as proposed for the tyrosine of other HDACs.…”

Crystal Structure of a Bacterial Class 2 Histone Deacetylase Homologue

“…This observation is supported by previous findings indicating a 100-fold weaker binding of inhibitors in the absence of the zinc ion. 17 At least in the case of CypX, a conformational flexibility of the ring moieties may also contribute to the less defined electron density. The two rings of Phe152 and Phe208, which line the active site channel, are in a co-planar orientation and could sandwich an inhibitor containing an aromatic ring system (Figure 1(c)).…”

“…they process 3-N-trifluoroacetyl-lysine-containing substrates. 17,25 FB188 HDAH activity is blocked by common HDAC inhibitors containing a hydroxamate group like SAHA (suberoylanilide hydroxamic acid), and also by typical class 2 specific inhibitors such as 3-[4-(3-(3-chlorophenyl)-3-oxo-1-propen-1-yl)-1-methyl-1H-pyrrol-2-yl]-Nhydroxy-2-propen-amide (3c published by Mai et al; 26 see Table 1). Known class 1 specific inhibitors like HC-toxin and chlamydocin (both inhibitors are epoxyketones), that do not inhibit class 2 enzymes like HDAC6, 27,28 also do not inhibit FB188 HDAH (Table 1).…”

“…[14][15][16] Accordingly, the bacterial histone deacetylase-like amidohydrolases (HDAHs) have also been proposed to act as protein deacetylases. 17 Based on sequence analysis HDACs have been divided into four groups and denoted as classes 1, 2, 0022-2836/$ -see front matter q 2005 Elsevier Ltd. All rights reserved.…”

A novel GaN‐based multiparameter sensor system for biochemical analysis

“…The measurement cycles are automated through a translation stage and a microscope camera. The enzyme HDAH can be produced easily in large quantities [9] and was chosen as a biochemical model because of its similarity to histone deacetylase (HDAC), a promising target in cancer therapy. The enzyme solution was diluted to 10 µg/ml and substrate solution to 83 µM using a weakly buffered solution of 500 µM BIS-TRIS propane, 100 µM citric acid, 1 mM NaCl, and 0.01 mg/ml bovine serum albumine at pH 8.6.…”