Purification, Cloning, Characterization and Essential Amino Acid Residues Analysis of a New iota-Carrageenase from Cellulophaga sp QY3
Author(s)
Type
Journal Article
Abstract
ι-Carrageenases belong to family 82 of glycoside hydrolases that degrade sulfated galactans in the red algae known as ι-carrageenans. The catalytic mechanism and some substrate-binding residues of family GH82 have been studied but the substrate recognition and binding mechanism of this family have not been fully elucidated. We report here the purification, cloning and characterization of a new ι-carrageenase CgiA_Ce from the marine bacterium Cellulophaga sp. QY3. CgiA_Ce was the most thermostable carrageenase described so far. It was most active at 50°C and pH 7.0 and retained more than 70% of the original activity after incubation at 50°C for 1 h at pH 7.0 or at pH 5.0–10.6 for 24 h. CgiA_Ce was an endo-type ι-carrageenase; it cleaved ι-carrageenan yielding neo-ι-carrabiose and neo-ι-carratetraose as the main end products, and neo-ι-carrahexaose was the minimum substrate. Sequence analysis and structure modeling showed that CgiA_Ce is indeed a new member of family GH82. Moreover, sequence analysis of ι-carrageenases revealed that the amino acid residues at subsites −1 and +1 were more conserved than those at other subsites. Site-directed mutagenesis followed by kinetic analysis identified three strictly conserved residues at subsites −1 and +1 of ι-carrageenases, G228, Y229 and R254 in CgiA_Ce, which played important roles for substrate binding. Furthermore, our results suggested that Y229 and R254 in CgiA_Ce interacted specifically with the sulfate groups of the sugar moieties located at subsites −1 and +1, shedding light on the mechanism of ι-carrageenan recognition in the family GH82.
Date Issued
2013-05-31
Date Acceptance
2013-04-16
Citation
PLOS ONE, 2013, 8 (5)
ISSN
1932-6203
Publisher
PUBLIC LIBRARY OF SCIENCE
Journal / Book Title
PLOS ONE
Volume
8
Issue
5
Copyright Statement
ß
2013 Ma et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
2013 Ma et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Identifier
http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000319799900076&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=1ba7043ffcc86c417c072aa74d649202
Subjects
Science & Technology
Multidisciplinary Sciences
Science & Technology - Other Topics
KAPPA-CARRAGEENASE
GLYCOSIDE HYDROLASES
PSEUDOALTEROMONAS-CARRAGEENOVORA
LAMBDA-CARRAGEENAN
STRUCTURAL BASIS
BETA-AGARASE
PROTEIN
FAMILY
OLIGOSACCHARIDES
DEGRADATION
Algal Proteins
Amino Acid Sequence
Binding Sites
Carrageenan
Glycoside Hydrolases
Hot Temperature
Kinetics
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Protein Binding
Rhodophyta
Substrate Specificity
MD Multidisciplinary
General Science & Technology
Publication Status
Published
Article Number
ARTN e64666