Photon Factory Activity Report 2016 #34 (2017)

BL-1A, AR-NW12A/2015G126

Structure Study of a Novel Type of Chitinase from Archaea
Yuichi Nishitani and Kunio Miki*
Graduate School of Science, Kyoto University, Kyoto, 606-8502, Japan
1 Introduction
Chitinase D (designated as Pc-ChiD) was found in a
hyperthermophilic archaeon Pyrococcus chitonophagus(previously described as Thermococcus chitonophagus),
isolated from media containing only chitin as carbon
source [1]. Pc-ChiD displays chitinase activity and isthermostable at temperatures up to 95˚C, raising its
potential for industrial use. Pc-ChiD has a secretion
signal peptide and two chitin-binding domains (ChBDs)
in the N-terminal domain. However, the C-terminal
domain shares no sequence similarity with previously
known saccharide-degrading enzymes, and does notcontain the DXDXE motif conserved in the glycosidehydrolase (GH) 18-family chitinases. To elucidate its
overall structure and reaction mechanism, we tried todetermine the first crystal structures of Pc-ChiD, both in
the ligand-free form and in complexes with substrates.
2 Experimental
We used Pc-ChiD(DBD), a truncated form of Pc-ChiD
which consists only of the C-terminal putative chitinasecatalytic domain based on primary structure and waspreviously shown to exhibit chitinase activity. The target
proteins were overexpressed with Escherichia coli. They
were purified with a heat treatment, an anion exchange
column, a hydrophobic interaction column, and a gel
filtration column. Crystals of the ligand-free form wereobtained using a reservoir solution containing (NH4)2SO4.
Crystals of the substrate-complex were obtained by theco-crystallization method using a reservoir solution
containing NH4H2PO4. X-ray diffraction data werecollected at the BL-1A and AR-NW12A beamlines at thePhoton Factory, and were processed with the HKL2000
package. The structures of Pc-ChiD(DBD) weredetermined by the single isomorphous replacement
method, using the platinum derivative.
3 Results and Discussion
We determined three types of Pc-ChiD crystal
structures, one ligand-free form and two substratecomplex forms, respectively. Structure analyses revealed
that Pc-ChiD(DBD) consists of a third ChBD (ChBD3),
which cannot be predicted from the amino acid sequence,
and a catalytic domain similar to those found in not theGH18 family but the GH23 family (Fig. 1). Based on thesimilarity with GH23-family chitinase, the catalytic
residues of Pc-ChiD were proposed and confirmed bymutagenesis analyses.
Sequence alignment in this family based on Dali server
results indicated that Pc-ChiD has a unique C-terminalextension. The C-terminal helix is positioned between

ChBD3 and the catalytic domain and fixes ChBD3 next tothe catalytic domain. Moreover, the Pc-ChiD structures incomplexes with ligands revealed that the two ligands arebound in a straight line, and two aromatic side chainsTrp391 (in ChBD3) and Tyr798 (in the C-terminalextension) are located at an extension of this line (Fig. 2).
These residues may contribute to the binding with thelong chitin chain. These findings indicate that the Cterminal extension functions not only for structure rigidityof the protein, but also for chitin-chain binding. Ourfindings reveal the structure of a unique archaeal
chitinase that is distinct from previously known membersof the GH23 family.

Fig. 1: Overall structure of Pc-ChiD(DBD).

Fig. 2: Proposed chitin-chain binding site.
References[1] A. Horiuchi et al., Appl. Environ. Microbiol. 82, 3554
(2016).
* miki@kuchem.kyoto-u.ac.jp