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...

A cell-free extract from Escherichia coli containing an E. coli biotin synthase that was expressed to approx. 1 % of soluble cell protein by cloning the E. coli bioB gene was used to investigate the biotin synthase reaction. The pH optimum was between 8 and 8n5, and the reaction velocity was dependent on the concentrations of dethiobiotin, cysteine, S-adenosylmethionine and asparagine. The catalytic-centre activity of the enzyme in itro was estimated to be 0n95 h −" , and each molecule of enzyme turned over less than one molecule of dethiobiotin, i.e. the enzyme was not acting catalytically. HPLC analysis of reaction mixtures revealed the presence of a compound with the characteristics of an intermediate : (1) it was labelled with "%C, and therefore derived from the ["%C]dethiobiotin substrate ; (2) it was present only in reaction mixtures containing biotin synthase ; (3) it was not derived from ["%C]biotin ; (4) $&S from [$&S]cystine was

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