Mechanism of enzymatic degradation of recalcitrant structuralpolysaccharide with crystalline structure
Takeshi WATANABE

Faculty of Agriculture, Niigata University
Research aims
Chitin is the second-most-abundant biopolymer in nature and is composed of
β-1,4-linked N-acetylglucosamine (GlcNAc). It is a common constituent of insect
exoskeletons, crustacean shells, and fungal cell walls. Chitin and its derivatives have
many potential applications owing to a multitude of useful functionalities. However,
enzymatic hydrolysis of chitin is particularly difficult because of a rigid crystallinestructure. To utilize this valuable and plentiful biomass resource, it is necessary tounderstand the mechanism of crystalline chitin hydrolysis that occurs naturally inbacterial chitinases. Recently, we have succeeded in observing chitinase molecules
hydrolyzing chitin chains on the surface of crystalline chitin using High-Speed Atomic
Force Microscopy (HS-AFM). This has added significant weight to a model ofcrystalline chitin hydrolysis by bacterial chitinase1) that was proposed by our groupseveral years ago2). We are now investigating:① Local structures in the chitinase molecule that are essential for crystalline chitinhydrolysis.
② The importance of CBP21 in chitin degradation and utilization, in addition tounderstanding its expression and physiological function in chitinolytic bacteria.

Methods① Investigating the local structure of the chitinase molecule, which is essential for
crystalline chitin hydrolysis
From our recent studies on chitinase, it is becoming apparent that aromatic aminoacid residues, both on the surface and inside the substrate-binding cleft, play a vital rolein crystalline chitin hydrolysis. To understand the details of their function, aromatic
amino acid residues, either on the surface or within the substrate-binding cleft, weresubstituted with other amino acid residues via site-directed mutagenesis. The effects ofthese substitutions were analyzed by HS-AFM and conventional enzymologicalanalysis.
② The importance of CBP21 in chitin degradation and utilization, in addition tounderstanding its expression and physiological function in chitinolytic bacteria
The chitin binding protein CBP21 from S.
marcescens has been reported to enhance chitin
degradation by chitinases. CBP21 is an oxidativeenzyme (lytic polysaccharide monooxygenase,LPMO) that generates new ends on chitin chainsat the surface of crystalline chitin. These newends enable chitinases to more easily bind andprocess chitin chains. Various chitinolytic bacteria

Fig 1 ． Processive hydrolysis model ofcrystalline β-chitin.

appear to possess proteins similar to CBP21. In order to elucidate the importance ofCBP21 in chitin utilization and its physiological function in chitinolytic bacteria, aCBP21 deletion mutant was constructed. Using this mutant, the effects that CBP21deletion had on the utilization of various type of chitin, bacterial growth, production ofchitinases, and pathogenicity in the insect host, were investigated.
Results① Investigating the local structure of the chitinase molecule, which is essential for
crystalline chitin hydrolysis
To elucidate the importance and function of aromatic amino acid residues exposed on
the surface of chitinase molecules, we focused on the two Tyr residues of ChiB. Tyr240,
most proximal to the substrate-binding cleft, and Tyr481, most distal to thesubstrate-binding cleft, were substituted with Trp. The effects on the binding andhydrolytic activities of the highly crystalline β-chitin microfibrils were investigated.

Substitution of either Tyr240 or Tyr481 by Trp did not significantly affect chitin-binding
activity. On the other hand, Tyr241→Trp mutation significantly decreased hydrolytic
activity against β-chitin microfibrils, indicating that the two aromatic residues possess
different functions. These results suggest that Tyr240 has additional roles in thedegradation of highly crystalline β-chitin, possibly by guiding a chitin chain into thecatalytic cleft.
To study the function of the aromatic amino acid residues within the substratebinding cleft, we focused on the Trp167 residue in ChiA. Trp167 was substituted withTyr, and the effect on binding and hydrolytic activity was examined. This mutation not
only affected the binding of ChiA to crystalline β-chitin microfibrils, but also thebinding of (GlcNAc)6. No significant differences were observed in any thermodynamic
binding parameter examined. Conversely, hydrolytic activity against highly crystalline
β-chitin microfibrils decreased significantly (approximately 30% of wild-type ChiA).
We predict that the Trp167→Tyr mutation reduces the rate at which the chitin chain
enters the substrate-binding cleft towards catalytic site.
② Importance of CBP21 in chitin degradation and utilization and understanding
its expression of physiological function in chitinolytic bacteria
Clearing zone formation by the CBP21 deletion mutant on agar plates containing
β-chitin microfibrils was significantly smaller and more
ambiguous compared to that of the wild-type strain. This
indicates that this protein is particularly important fordegradation and utilization of β-chitin microfibrils. Inaddition, deletion of CBP21 had a more severe effect on thedegradation and utilization of chitin fragment flakes larger
than fine powder. Unexpectedly, deletion of CBP21 did Fig ２．Clearing zone of β-chitin.
not affect pathogenicity in the insect host.

Wild type (top), CBP-deficientmutant (left), and complemented
strain (right).

Conclusions
In the processive hydrolysis of crystalline chitin, aromatic amino acid residues play
an important role. The functions of individual residue are very different from each other,and depend on the position and type of aromatic side chain. CBP21 enhances
degradation of chitin by chitinases via a sophisticated mechanism. Our observation thatCBP21 acts more effectively on chitin in a more native state suggests that the enzyme
plays a critical role in recycling chitin in nature.

References
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Sakuda, S., Ando, T., Watanabe, T. and Samejima, M. (2014) Two-way traffic of glycosidehydrolase family 18 processive chitinases on crystalline chitin. Nature Communications. 5,Article number: 3975.
2) Uchiyama, T., Katouno, F., Nikaidou, N., Nonaka, T., Sugiyama, J. and Watanabe, T. (2001)
Roles of the exposed aromatic residues in crystalline chitin hydrolysis by chitinase A fromSerratia marcescens 2170. J. Biol. Chem. 276(44):41343-41349.
3) Sugimoto, H., Nakamura, K., Nishino, Y., Idezawa, Y., Fujinuma, Y., Suzuki, K., andWatanabe, T. (2016) Differences in the roles of the two surface-exposed tyrosine residues,
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