Structure, activity, and stability of metagenome‐derived glycoside hydrolase family 9 endoglucanase with an N‐terminal Ig‐like domain. (13th January 2015)

• Record Type: Journal Article
• Authors: Okano, Hiroyuki; Kanaya, Eiko; Ozaki, Masashi; Angkawidjaja, Clement; Kanaya, Shigenori
• Is Part Of: Protein science. Volume 24:Number 3(2015:Mar.)
• Page Start: 408
• Page End: 419
• Publication Date: 2015-01-13
• Subjects: Proteins -- Periodicals; 572.6
• DOI: 10.1002/pro.2632
• Languages: English
• ISSNs: 0961-8368

Abstract

<abstract abstract-type="main"> <title>Abstract</title> <p>A metagenome‐derived glycoside hydrolase family 9 enzyme with an N‐terminal immunoglobulin‐like (Ig‐like) domain, leaf‐branch compost (LC)‐CelG, was characterized and its crystal structure was determined. LC‐CelG did not hydrolyze <italic>p</italic>‐nitrophenyl cellobioside but hydrolyzed CM‐cellulose, indicating that it is endoglucanase. LC‐CelG exhibited the highest activity at 70°C and >80% of the maximal activity at a broad pH range of 5–9. Its denaturation temperature was 81.4°C, indicating that LC‐CelG is a thermostable enzyme. The structure of LC‐CelG resembles those of CelD from <italic>Clostridium thermocellum</italic> (CtCelD), Cel9A from <italic>Alicyclobacillus acidocaldarius</italic> (AaCel9A), and cellobiohydrolase CbhA from <italic>C. thermocellum</italic> (CtCbhA), which show relatively low (29–31%) amino acid sequence identities to LC‐CelG. Three acidic active site residues are conserved as Asp194, Asp197, and Glu558 in LC‐CelG. Ten of the thirteen residues that form the substrate binding pocket of AaCel9A are conserved in LC‐CelG. Removal of the Ig‐like domain reduced the activity and stability of LC‐CelG by 100‐fold and 6.3°C, respectively. Removal of the Gln40‐ and Asp99‐mediated interactions between the Ig‐like and catalytic domains destabilized LC‐CelG by 5.0°C without significantly affecting its activity. These results suggest that the Ig‐like domain contributes to the stabilization of LC‐CelG mainly due to the Gln40‐ and Asp99‐mediated interactions. Because the LC‐CelG derivative lacking the Ig‐like domain accumulated in <italic>Escherichia coli</italic> cells mostly in an insoluble form and this derivative accumulated in a soluble form exhibited very weak activity, the Ig‐like domain may be required to make the conformation of the active site functional and prevent aggregation of the catalytic domain.</p> </abstract>