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13 pages, 3129 KB

Open AccessArticle

A Simplified Method for Evaluating Chitin-Binding Activity Applied to YKL-40 (HC-gp39, CHI3L1) and Chitotriosidase

by Keita Suzuki, Hidetoshi Suzuki, Ami Tanaka, Miwa Tanaka, Kairi Takase, Hiromu Takei, Tomoki Kanaizumi, Kazuaki Okawa, Peter O. Bauer and Fumitaka Oyama

Molecules 2025, 30(1), 19; https://doi.org/10.3390/molecules30010019 - 25 Dec 2024

Cited by 1 | Viewed by 2310

Abstract

YKL-40 is structurally similar to chitotriosidase (CHIT1), an active chitinase, but it lacks chitin-degrading activity while retaining chitin-binding capability. Elevated YKL-40 levels are associated with inflammatory diseases and cancers, making it a valuable biomarker. We previously reported that the W69T substitution in YKL-40 significantly reduces its chitin-binding affinity, identifying W69 as a crucial binding site. In this study, we establish a novel chitin-binding affinity evaluation method using a three-step buffer system to assess the binding strength and specificity of chitin-binding proteins and apply it to characterize YKL-40’s binding mechanism. Our findings confirm that YKL-40, through its key residue W69, exhibits highly specific and robust affinity to chitin. Unlike CHIT1, which has both a catalytic domain (CatD) and a chitin-binding domain (CBD) that allow for diverse binding and degradation activities, YKL-40 lacks a CBD and is specialized for specific chitin recognition without degrading it. Comparative analysis with YKL-39, which does not contain a corresponding W69 residue, highlights the unique role of this residue in YKL-40’s chitin-binding activity that is potentially linked to immune and inflammatory responses. Our evaluation method clarifies YKL-40’s binding properties and provides a versatile approach applicable to other chitin-binding proteins. Full article

(This article belongs to the Section Bioorganic Chemistry)

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21 pages, 505 KB

Open AccessArticle

Integral Characteristic of Complex Catalytic Reaction Accompanied by Deactivation

by Zoë Gromotka, Gregory Yablonsky, Nickolay Ostrovskii and Denis Constales

Catalysts 2022, 12(10), 1283; https://doi.org/10.3390/catal12101283 - 20 Oct 2022

Cited by 3 | Viewed by 2301

Abstract

New theoretical relationships for a complex catalytic reaction accompanied by deactivation are obtained, using as an example the two-step catalytic mechanism (Temkin–Boudart mechanism) with irreversible reactions and irreversible deactivation. In the domain of small concentrations, [...] Read more.

A_{\lim} = N_{S} \frac{k_{1} C_{A}}{k_{d}},

where

A_{\lim}

is the limit of the integral consumption of the gas substance,

N_{S}

is the number of active sites per unit of catalyst surface;

k_{1}

and

k_{d}

, are kinetic coefficients which relate to two reactions which compete for the free active site Z.

C_{A}

is the gas concentration. One reaction belongs to the catalytic cycle. The other reaction with kinetic coefficient

k_{d}

is irreversible deactivation. The catalyst lifetime,

τ_{cat} = \frac{1}{C_{Z}^{'}} \frac{1}{k_{d}}

, where

C_{Z}^{'}

is the dimensionless steady-state concentration of free active sites. The main conclusion was formulated as follows: the catalyst lifetime can be enhanced by decreasing the steady-state (quasi-steady-state) concentration of free active sites. In some domains of parameters, it can also be achieved by increasing the steady-state (quasi-steady-state) reaction rate of the fresh catalyst. We can express this conclusion as follows: under some conditions, an elevated fresh catalyst activity protects the catalyst from deactivation. These theoretical results are illustrated with the use of computer simulations. Full article

(This article belongs to the Section Catalytic Materials)

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16 pages, 1450 KB

Open AccessArticle

Chitinase Chit62J4 Essential for Chitin Processing by Human Microbiome Bacterium Clostridium paraputrificum J4

by Jan Dohnálek, Jarmila Dušková, Galina Tishchenko, Petr Kolenko, Tereza Skálová, Petr Novák, Karla Fejfarová and Jiří Šimůnek

Molecules 2021, 26(19), 5978; https://doi.org/10.3390/molecules26195978 - 2 Oct 2021

Cited by 8 | Viewed by 3823

Abstract

Commensal bacterium Clostridium paraputrificum J4 produces several extracellular chitinolytic enzymes including a 62 kDa chitinase Chit62J4 active toward 4-nitrophenyl N,N′-diacetyl-β-d-chitobioside (pNGG). We characterized the crude enzyme from bacterial culture fluid, recombinant enzyme rChit62J4, and its catalytic domain rChit62J4cat. This major chitinase, securing nutrition of the bacterium in the human intestinal tract when supplied with chitin, has a pH optimum of 5.5 and processes pNGG with K_m = 0.24 mM and k_cat = 30.0 s⁻¹. Sequence comparison of the amino acid sequence of Chit62J4, determined during bacterial genome sequencing, characterizes the enzyme as a family 18 glycosyl hydrolase with a four-domain structure. The catalytic domain has the typical TIM barrel structure and the accessory domains—2x Fn3/Big3 and a carbohydrate binding module—that likely supports enzyme activity on chitin fibers. The catalytic domain is highly homologous to a single-domain chitinase of Bacillus cereus NCTU2. However, the catalytic profiles significantly differ between the two enzymes despite almost identical catalytic sites. The shift of pI and pH optimum of the commensal enzyme toward acidic values compared to the soil bacterium is the likely environmental adaptation that provides C. paraputrificum J4 a competitive advantage over other commensal bacteria. Full article

(This article belongs to the Special Issue Recent Advances in Carbohydrate-Active Enzymes)

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15 pages, 2436 KB

Open AccessArticle

Functional Properties of the Catalytic Domain of Mouse Acidic Mammalian Chitinase Expressed in Escherichia coli

by Akinori Kashimura, Masahiro Kimura, Kazuaki Okawa, Hirotaka Suzuki, Atsushi Ukita, Satoshi Wakita, Kana Okazaki, Misa Ohno, Peter O. Bauer, Masayoshi Sakaguchi, Yasusato Sugahara and Fumitaka Oyama

Int. J. Mol. Sci. 2015, 16(2), 4028-4042; https://doi.org/10.3390/ijms16024028 - 13 Feb 2015

Cited by 24 | Viewed by 7386

Abstract

Mouse acidic mammalian chitinase (AMCase) plays important physiological roles in defense and nutrition. AMCase is composed of an N-terminal catalytic domain (CatD) and a C-terminal chitin-binding domain (CBD). We expressed CatD of mouse AMCase as a recombinant fusion protein with Protein A and V5-His in Escherichia coli (Protein A-CatD-V5-His), evaluated its functional properties and compared them to the full-length AMCase (Protein A-AMCase-V5-His). Under our experimental conditions, the chitinolytic activity of both proteins against 4-nitrophenyl N,N'-diacetyl-β-d-chitobioside was equivalent with regard to their specific enzymatic activities, optimal pH and temperature as well as to the pH and temperature stability. CatD bound to chitin beads and cleaved the N-acetylglucosamine hexamer, colloidal and crystalline chitin as well as the shrimp shell, and released primarily N,N'-diacetylchitobiose fragments at pH 2.0. These results indicate that the primary structure of CatD is sufficient to form a proper tertiary structure required for chitinolytic activity, recognize chitin substrates and degrade them in the absence of a CBD. Our recombinant proteins can be used for further studies evaluating pathophysiological roles of AMCase in different diseases. Full article

(This article belongs to the Section Biochemistry)

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