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J. Biol. Chem., Vol. 283, Issue 46, 31551-31558, November 14, 2008

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Biochemical and Structural Characterization of the Intracellular Mannanase AaManA of Alicyclobacillus acidocaldarius Reveals a Novel Glycoside Hydrolase Family Belonging to Clan GH-A^*

Yueling Zhang, Jiansong Ju, Hao Peng, Feng Gao, Cheng Zhou, Yan Zeng, Yanfen Xue, Yin Li, Bernard Henrissat^¶, George F. Gao, and Yanhe Ma¹

From the State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China, the Graduate School, Chinese Academy of Sciences, Beijing 100049, China, and ^¶Architecture et Fonction des Macromolécules Biologiques, UMR6098, CNRS, and Universités d'Aix-Marseille I and II, 163 Avenue de Luminy, 13288 Marseille, France

An intracellular mannanase was identified from the thermoacidophile Alicyclobacillus acidocaldarius Tc-12-31. This enzyme is particularly interesting, because it shows no significant sequence similarity to any known glycoside hydrolase. Gene cloning, biochemical characterization, and structural studies of this novel mannanase are reported in this paper. The gene consists of 963 bp and encodes a 320-amino acid protein, AaManA. Based on its substrate specificity and product profile, AaManA is classified as an endo-β-1,4-mannanase that is capable of transglycosylation. Kinetic analysis studies revealed that the enzyme required at least five subsites for efficient hydrolysis. The crystal structure at 1.9Å resolution showed that AaManA adopted a (β/)₈-barrel fold. Two catalytic residues were identified: Glu¹⁵¹ at the C terminus of β-stand β4 and Glu²³¹ at the C terminus of β7. Based on the structure of the enzyme and evidence of its transglycosylation activity, AaManA is placed in clan GH-A. Superpositioning of its structure with that of other clan GH-A enzymes revealed that six of the eight GH-A key residues were functionally conserved in AaManA, with the exceptions being residues Thr⁹⁵ and Cys¹⁵⁰. We propose a model of substrate binding in AaManA in which Glu²⁸² interacts with the axial OH-C(2) in–2 subsites. Based on sequence comparisons, the enzyme was assigned to a new glycoside hydrolase family (GH113) that belongs to clan GH-A.

Received for publication, May 5, 2008 , and in revised form, August 15, 2008.

^* This study was supported by Chinese Academy of Sciences Knowledge Innovation Program Grant KSCX2-SW-33, National Natural Science Foundation of China Grant 30621005, and Ministry of Sciences and Technology of China (973 programs) Grants 2003CB716001 and 2007CB707801. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4 and Table S1.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) DQ838045 [GenBank] and DQ680160.

The atomic coordinates and structure factors (code 3CIV) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

¹ To whom correspondence should be addressed: Institute of Microbiology, Chinese Academy of Sciences, A3 Datun Rd., Chaoyang District, Beijing 100101, China. Tel.: 86-10-64807590; Fax: 86-10-64807616; E-mail: mayanhe{at}im.ac.cn.

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