Structural studies of a thermophilic esterase from a new Planctomycetes species, Thermogutta terrifontis. (17th June 2015)

• Record Type: Journal Article
• Authors: Sayer, Christopher; Isupov, Michail N.; Bonch‐Osmolovskaya, Elizaveta; Littlechild, Jennifer A.
• Is Part Of: FEBS journal. Volume 282:Number 15(2015)
• Page Start: 2846
• Page End: 2857
• Publication Date: 2015-06-17
• Subjects: Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.13326
• Languages: English
• ISSNs: 1742-464X

Abstract

<abstract abstract-type="main" id="febs13326-abs-0001"> <title> <x xml:space="preserve">Abstract</x> </title> <sec id="febs13326-sec-0001" sec-type="section"> <p> <italic>Thermogutta terrifontis</italic> esterase (TtEst), a carboxyl esterase identified in the novel thermophilic bacterium <italic>T. terrifontis</italic> from the phylum Planctomycetes, has been cloned and over‐expressed in <italic>Escherichia coli</italic>. The enzyme has been characterized biochemically and shown to have activity towards small <italic>p</italic>‐nitrophenyl (<italic>p</italic>NP) carboxylic esters, with optimal activity for <italic>p</italic>NP‐propionate. The enzyme retained 95% activity after incubation for 1 h at 80 °C. The crystal structures of the native TtEst and its complexes with the substrate analogue <sc>d</sc>‐malate and the product acetate have been determined to high resolution. The bound ligands have allowed the identification of the carboxyl and alcohol binding pockets in the enzyme active site. Comparison of TtEst with structurally related enzymes provides insight into how differences in their catalytic activity can be rationalized based upon the properties of the amino acid residues in their active site pockets. The mutant enzymes L37A and L251A have been constructed to extend the substrate range of TtEst towards the larger butyrate and valerate <italic>p</italic>NP‐esters. These mutant enzymes have also shown a significant increase in activity towards acetate and propionate <italic>p</italic>NP esters. A crystal structure of the L37A mutant was determined with the butyrate product bound in the carboxyl pocket of the active site. The mutant structure shows an expansion of the pocket that binds the substrate carboxyl group, which is consistent with the observed increase in activity towards <italic>p</italic>NP‐butyrate.</p> </sec> <sec id="febs13326-sec-0002" sec-type="section"> <title>Database</title> <p>The GenBank sequence accession number for the <italic>Thermogutta terrifontis</italic> esterase is KR002593. The protein structures for <italic>T. terrifontis</italic> esterase and their complexes have been deposited in the Protein Data Bank with codes: <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4UHC" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">4UHC</ext-link> (native), <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4UHD" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">4UHD</ext-link> (acetate bound), <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4UHE" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">4UHE</ext-link> (malate bound) and <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4UHF" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">4UHF</ext-link> (L37A mutant with butyrate bound).</p> </sec> </abstract>