Expression from engineered Escherichia coli chromosome and crystallographic study of archaeal N, N′‐diacetylchitobiose deacetylase. (28th April 2014)

• Record Type: Journal Article
• Authors: Mine, Shouhei; Niiyama, Mayumi; Hashimoto, Wakana; Ikegami, Takahisa; Koma, Daisuke; Ohmoto, Takashi; Fukuda, Yohta; Inoue, Tsuyoshi; Abe, Yoshito; Ueda, Tadashi; Morita, Junji; Uegaki, Koichi; Nakamura, Tsutomu
• Is Part Of: FEBS journal. Volume 281:Number 11(2014)
• Page Start: 2584
• Page End: 2596
• Publication Date: 2014-04-28
• Subjects: Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.12805
• Languages: English
• ISSNs: 1742-464X

Abstract

<abstract abstract-type="main" id="febs12805-abs-0001"> <title> <x xml:space="preserve">Abstract</x> </title> <sec id="febs12805-sec-0001" sec-type="section"> <p>In order to develop a structure‐based understanding of the chitinolytic pathway in hyperthermophilic <italic>Pyrococcus</italic> species, we performed crystallographic studies on <italic>N</italic>, <italic>N</italic>′‐diacetylchitobiose deacetylases (Dacs) from <italic>Pyrococcus horikoshii</italic> (Ph‐Dac) and <italic>Pyrococcus furiosus</italic> (Pf‐Dac). Neither Ph‐Dac nor Pf‐Dac was expressed in the soluble fraction of <italic>Escherichia coli</italic> harboring the expression plasmid. However, insertion of the target genes into the chromosome of <italic>E. coli</italic> yielded the soluble recombinant protein. The purified <italic>Pyrococcus</italic> Dacs were active and thermostable up to 85 °C. The crystal structures of Ph‐Dac and Pf‐Dac were determined at resolutions of 2.0 Å and 1.54 Å, respectively. The <italic>Pyrococcus</italic> Dac forms a hexamer composed of two trimers. These Dacs are characterized by an intermolecular cleft, which is formed by two polypeptides in the trimeric assembly. In Ph‐Dac, catalytic Zn situated at the end of the cleft is coordinated by three side chain ligands from His44, Asp47, and His155, and by a phosphate ion derived from the crystallization reservoir solution. We considered that the bound phosphate mimicked the tetrahedral oxyanion, which is an intermediate of hydrolysis of the N‐acetyl group, and proposed an appropriate reaction mechanism. In the proposed mechanism, the N<sup>ε</sup> atom of His264 (from the adjacent polypeptide in the Ph‐Dac sequence) is directly involved in the stabilization of the oxyanion intermediate. Mutation analysis also indicated that His264 was essential to the catalysis. These factors give the archaeal Dacs an unprecedented active site architecture a Zn‐dependent deacetylases.</p> </sec> <sec id="febs12805-sec-0102" sec-type="section"> <title>Database</title> <p>Structural data are available in the Protein Data Bank database under accession numbers <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=3WL3" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">3WL3</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=3WL4" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">3WL4</ext-link>, and <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=3WE7" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">3WE7</ext-link>.</p> </sec> <sec id="febs12805-sec-0002" sec-type="section"> <title>Structured digital abstract</title> <p> <ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/O58092" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">Ph-Dac</ext-link> and <ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/O58092" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">Ph-Dac</ext-link> <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">bind</ext-link> by <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">x-ray crystallography</ext-link> (<ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9351487" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">View interaction</ext-link>)</p> <p> <ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/Q8U3R6" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">Pf-Dac</ext-link> and <ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/Q8U3R6" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">Pf-Dac</ext-link> <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">bind</ext-link> by <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">x-ray crystallography</ext-link> (<ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9351510" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">View interaction</ext-link>)</p> </sec> </abstract>