Crystal structure of Grimontia hollisae collagenase provides insights into its novel substrate specificity toward collagen

Ikeuchi, Takeaki; Yasumoto, Mizuki; Takita, Teisuke; Tanaka, Keisuke; Kusubata, Masashi; Hayashida, Osamu; Hattori, Shunji; Mizutani, Kimihiko; Mikami, Bunzo; Yasukawa, Kiyoshi

doi:10.1016/j.jbc.2022.102109

Cited by 4 publications

(23 citation statements)

References 39 publications

(75 reference statements)

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“…The collected diffraction data were processed with XDS [18]. Molecular replacement was conducted using the structure of ligand-free Ghcol [17] as a search model (PDB, 7WSS). Structure refinement was conducted using COOT [19] and PHENIX [20].…”

Section: Crystallization and Structural Determinationmentioning

confidence: 99%

“…Expression plasmids for Ghcol variants were constructed by site-directed mutagenesis using the expression plasmid for wild-type Ghcol, pNY326-Col2 (Fig. S2) [16,17], as a template and Brevibacillus as a host. The primer sequences are shown in Table S1.…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…Expression and purification were carried out as described previously [16,17] with modifications. Briefly, each expression plasmid was transfected into Brevibacillus cells, and the cells were transferred into MTNm plates containing 50 μgÁmL À1 neomycin and stored at 37 °C for 16 h. One colony was inoculated in 2SY medium (5 mL) containing 50 μgÁmL À1 neomycin and 1 mM CaCl 2 and cultured at 30 °C with shaking for 16 h. The culture was added to 500 mL of 2SY medium containing 50 μgÁmL À1 neomycin and 1 mM CaCl 2 in a 1-L flask and incubated at 30 °C with shaking for 48 h. After centrifugation of the culture at 12 000 g for 15 min, and the supernatant was collected.…”

Section: Expression and Purification Of Ghcolmentioning

confidence: 99%

“…The activity was measured as described previously [14,16,17] with modifications. Briefly, substrate solution was prepared by mixing equal volumes of 0.1% FITClabeled type I collagen in 5 mM acetic acid and neutralizing solution [0.1 M Tris-HCl buffer (pH 7.5), 10 mM CaCl 2 , 400 mM NaCl].…”

Section: Hydrolysis Of Fitc-collagenmentioning

confidence: 99%

“…Recombinant Ghcol was expressed in Brevibacillus choshinensis and purified from the supernatants [12]. It exhibited higher collagendegrading activity than C. histolyticum collagenases [13][14][15][16][17], suggesting its potential use in industry.…”

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confidence: 99%

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Insights into the catalytic mechanism of Grimontia hollisae collagenase through structural and mutational analyses

Ueshima,

Yasumoto,

Kitagawa

et al. 2023

FEBS Letters

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Grimontia hollisae collagenase (Ghcol) exhibits high collagen‐degrading activity. To explore its catalytic mechanism, its substrate (Gly‐Pro‐Hyp‐Gly‐Pro‐Hyp, GPOGPO)‐complexed crystal structure was determined at 2.0 Å resolution. A water molecule was observed near the active‐site zinc ion. Since this water was not observed in the product (GPO)‐complexed Ghcol, it was hypothesized that the GPOGPO‐complexed Ghcol structure reflects a Michaelis complex, providing a structural basis for understanding the catalytic mechanism. Analyses of the active‐site geometry and site‐directed mutagenesis of the active‐site tyrosine residues revealed that Glu493 and Tyr564 were essential for catalysis, suggesting that Glu493 functions as an acid and base catalyst while Tyr564 stabilizes the tetrahedral complex in the transition state. These results shed light on the catalytic mechanism of bacterial collagenase.

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Section: Crystallization and Structural Determinationmentioning

confidence: 99%

Section: Plasmid Constructionmentioning

confidence: 99%

Section: Expression and Purification Of Ghcolmentioning

confidence: 99%

Section: Hydrolysis Of Fitc-collagenmentioning

confidence: 99%

mentioning

confidence: 99%

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Insights into the catalytic mechanism of Grimontia hollisae collagenase through structural and mutational analyses

Ueshima,

Yasumoto,

Kitagawa

et al. 2023

FEBS Letters

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A conserved strategy to attack collagen: The activator domain in bacterial collagenases unwinds triple-helical collagen

Serwanja,

Wieland,

Haubenhofer

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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Bacterial collagenases are important virulence factors, secreted by several pathogenic Clostridium , Bacillus , Spirochaetes , and Vibrio species. Yet, the mechanism by which these enzymes cleave collagen is not well understood. Based on biochemical and mutational studies we reveal that collagenase G (ColG) from Hathewaya histolytica recognizes and processes collagen substrates differently depending on their nature (fibrillar vs. soluble collagen); distinct dynamic interactions between the activator and peptidase domain are required based on the substrate type. Using biochemical and circular dichroism studies, we identify the presumed noncatalytic activator domain as the single-domain triple helicase that unwinds collagen locally, transiently, and reversibly.

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The activator domain of bacterial collagenases drives collagen recognition, unwinding and processing

Serwanja,

Wieland,

Haubenhofer

et al. 2023

Preprint

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Collagens form the resilient backbone of the extracellular matrix in mammals. Only few proteases are able to digest triple-helical collagen. Clostridial collagenases can efficiently process collagen. However, little is known about the mechanism of bacterial collagenolyis of either soluble collagen or the multi-hierarchically assembled, insoluble collagen fibers. Here we present a functional analysis of the distinct roles of the individual domains of collagenase G (ColG) from Hathewaya histolytica. A broad array of biochemical, biophysical, and enzymatic data consistently revealed unexpected synergistic and antagonistic interactions between the activator, peptidase and collagen-binding domains. We found the non-catalytic activator domain to act as a master regulator, coordinating the complex interactions to specifically recognize and process the diverse physiological substrates. The results presented here enable multiple applications such as the engineering of collagenase variants with selectivity for defined substrate states.

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Crystal structure of Grimontia hollisae collagenase provides insights into its novel substrate specificity toward collagen

Cited by 4 publications

References 39 publications

Insights into the catalytic mechanism of Grimontia hollisae collagenase through structural and mutational analyses

Insights into the catalytic mechanism of Grimontia hollisae collagenase through structural and mutational analyses

A conserved strategy to attack collagen: The activator domain in bacterial collagenases unwinds triple-helical collagen

The activator domain of bacterial collagenases drives collagen recognition, unwinding and processing

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