A new amidohydrolase from Bordetella or Alcaligenes strain FB188 with similarities to histone deacetylases
A new amidohydrolase from Bordetella or Alcaligenes strain FB188 with similarities to histone deacetylases
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DOI：
10.1128/jb.186.8.2328-2339.2004
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发表时间：
2004-04-01
影响因子：
3.2
通讯作者：
Schwienhorst, A
中科院分区：
文献类型：
--
作者：
Hildmann, C;Ninkovic, M;Schwienhorst, A
采用简并引物PCR结合反向PCR技术，从博德特氏菌属或产碱杆菌属菌株FB 188（DSM 11172）中克隆了编码组蛋白脱乙酰酶（HDAC）样酰胺水解酶（HDAH）的全长基因，并最终在大肠杆菌中表达。表达的酶进行了生物化学表征，并发现类似于天然酶的所有性能检查。核苷酸序列分析揭示了一个1，110 bp的开放阅读框架，其编码理论分子量为39 kDa的多肽。有趣的是，肽测序揭示了成熟野生型酶中缺乏N-末端甲硫氨酸，推测是由于甲硫氨酰氨基肽酶的作用。序列数据库搜索表明，新的酰胺水解酶属于HDAC超家族，最接近的同系物被发现在亚家族分配的乙酰多胺酰胺水解酶（APAH）。APAH亚家族包括来自诸如铜绿假单胞菌、闪烁古生球菌和放线菌枝菌（Mycoplana ramosa）（以前称为M. bullata）。然而，FBI 88 HDAH在催化乙酰多胺的脱乙酰化中仅具有中等活性。事实上，FB 188 HDAH在标准HDAC测定中表现出显著的活性，并且被已知的HDAC抑制剂如阿司他汀A和辛二酰苯胺异羟肟酸（SAHA）抑制。多项证据表明，FB 188 HDAH与1类和2类HDAC非常相似，并且在活性位点含有Zn 2+离子，这对催化活性有显着贡献。最初的生物技术应用表明，广泛的底物谱和宽的最佳pH范围是使用来自博德特氏菌/产碱杆菌菌株FB 188的新HDAH作为技术生物转化中的生物催化剂的极好标准，属于人免疫缺陷病毒逆转录酶抑制剂合成的范围。
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 FBI 88 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 Zn2+ 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.