Molecular Analysis of the Gene Encoding a Novel Chitin-Binding Protease from
            <i>Alteromonas</i>
            sp. Strain O-7 and Its Role in the Chitinolytic System

Miyamoto, Katsushiro; Nukui, Eiji; Itoh, Hiroyuki; Sato, Takaaki; Kobayashi, Takeshi; Imada, Chiaki; Watanabe, Etsuo; Inamori, Yoshihiko; Tsujibo, Hiroshi

doi:10.1128/jb.184.7.1865-1872.2002

Cited by 27 publications

(27 citation statements)

References 43 publications

(18 reference statements)

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“…In addition to cell surface proteins, the PKD domain is found in many biopolymer hydrolases, such as chitinases (4,5), celluloses (6), and proteases (7)(8)(9), suggesting that it may play an important role in biopolymer degradation. The structures of three PKD domains have been solved, which show that, though their sequences are different, they all adopt a ␤-helix fold and a conserved sequence area with two Trp residues in the hydrophobic core (2,3).…”

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confidence: 99%

Mechanistic Insight into the Function of the C-terminal PKD Domain of the Collagenolytic Serine Protease Deseasin MCP-01 from Deep Sea Pseudoalteromonas sp. SM9913

Wang

Zhao

et al. 2010

Journal of Biological Chemistry

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Deseasin MCP-01 is a bacterial collagenolytic serine protease. Its catalytic domain alone can degrade collagen, and its C-terminal PKD domain is a collagen-binding domain (CBD) that can improve the collagenolytic efficiency of the catalytic domain by an unknown mechanism. Here, scanning electron microscopy (SEM), atomic force microscopy (AFM), zeta potential, and circular dichroism spectroscopy were used to clarify the functional mechanism of the PKD domain in MCP-01 collagenolysis. The PKD domain observably swelled insoluble collagen. Its collagenswelling ability and its improvement to the collagenolysis of the catalytic domain are both temperature-dependent. SEM observation showed the PKD domain swelled collagen fascicles with an increase of their diameter from 5.3 m to 8.8 m after 1 h of treatment, and the fibrils forming the fascicles were dispersed. AFM observation directly showed that the PKD domain bound collagen, swelled the microfibrils, and exposed the monomers. The PKD mutant W36A neither bound collagen nor disturbed its structure. Zeta potential results demonstrated that PKD treatment increased the net positive charges of the collagen surface. PKD treatment caused no change in the content or the thermostability of the collagen triple helix. Furthermore, the PKD-treated collagen could not be degraded by gelatinase. Therefore, though the triple helix monomers were exposed, the PKD domain could not unwind the collagen triple helix. Our study reveals the functional mechanism of the PKD domain of the collagenolytic serine protease MCP-01 in collagen degradation, which is distinct from that of the CBDs of mammalian matrix metalloproteases. The polycystic kidney disease (PKD)3 domain is an 80 -90-amino acid module originally found in the human PKD1 gene encoding the cell surface glycoprotein polycystin-1 (1), and later, in many surface layer proteins of archaebacteria (2, 3). In addition to cell surface proteins, the PKD domain is found in many biopolymer hydrolases, such as chitinases (4, 5), celluloses (6), and proteases (7-9), suggesting that it may play an important role in biopolymer degradation. The structures of three PKD domains have been solved, which show that, though their sequences are different, they all adopt a ␤-helix fold and a conserved sequence area with two Trp residues in the hydrophobic core (2, 3). However, the functional mechanism of the PKD domain in biopolymer hydrolases is largely unknown, except that the PKD domains in chitinase ChiA and collagenolytic serine protease deseasin MCP-01 are both reported to function as a binding domain (5, 10). Genome sequence analysis and experimental results show that the PKD domain is common in the hydrolases of marine heterotrophic bacteria. For example, Gramella forsetii has 14 exported proteins with PKD domains (11). Chitin-binding protease AprIV, deseasin MCP-01, and many other marine deseasins contain one or two PKD domains (5, 9). Therefore, elucidating the function and the mechanism of action of the PKD domains in these hydrolases would hav...

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confidence: 99%

Mechanistic Insight into the Function of the C-terminal PKD Domain of the Collagenolytic Serine Protease Deseasin MCP-01 from Deep Sea Pseudoalteromonas sp. SM9913

Wang

Zhao

et al. 2010

Journal of Biological Chemistry

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“…strain O-7 as a common structural unit. We have already demonstrated that the type 3 ChtBD strongly binds ␣-and ␤-chitin and is essential for efficient hydrolysis of insoluble chitin (21,37,41). These results indicate that the type 3 ChtBD of ChiB directly participates in the binding to chitin molecules and plays an important role in the hydrolysis of the insoluble substrate.…”

Section: Discussionmentioning

confidence: 84%

“…strain O-7 is an efficient degrader of chitin in the marine environment (33). We have already shown that the chitinolytic system of the strain consists of three chitinases (ChiA, ChiB, and ChiC), three ␤-N-acetylglucosaminidases (GlcNAcase A, GlcNAcase B, and GlcNAcase C), a chitinaselike enzyme (ChiD), a transglycosylative enzyme (Hex99), a chitin-binding protein (Cbp1), and a chitin-binding protease (AprIV) (21,(34)(35)(36)(37)(38)(39)41). In this paper, we report that ChiB is a modular enzyme with a high level of catalytic activity at low temperatures compared with ChiA and ChiC.…”

Section: Discussionmentioning

confidence: 99%

“…The membrane was incubated for 1 h at room temperature with anti-ChiB polyclonal rabbit anti- serum diluted to 1:1,000 in phosphate-buffered saline containing 2.0% skim milk (Difco). Bound antibody was detected as described previously (21). Real-time quantitative PCR analysis of the chiB transcript.…”

Section: Methodsmentioning

confidence: 99%

“…This strain produces at least three chitinases (ChiA, ChiB, and ChiC), a chitinase-like enzyme (ChiD), three ␤-N-acetylglucosaminidases (GlcNAcases A, B, and C), a transglycosylative enzyme (Hex99), a chitin-binding protein (Cbp1), and a chitin-binding protease (AprIV) in the presence of chitin. We have cloned and sequenced all of the genes involved in the chitin degradation of the strain except the chiB gene (21,(34)(35)(36)(37)(38)(39)41). Furthermore, they have been expressed in Escherichia coli and the biochemical properties of each recombinant protein have been investigated.…”

mentioning

confidence: 99%

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Molecular Analysis of the Gene Encoding a Novel Cold-Adapted Chitinase (ChiB) from a Marine Bacterium, Alteromonas sp. Strain O-7

et al. 2003

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The chitinase B (ChiB) secreted by Alteromonas sp. strain O-7 was purified, and the corresponding gene (chiB) was cloned and sequenced. The open reading frame of the chiB gene encodes a protein of 850 amino acids with a calculated molecular mass of 90,223 Da. ChiB is a modular enzyme consisting of two reiterated domains and a catalytic domain belonging to chitinase family 18. The reiterated domains are composed of chitin-binding domain (ChtBD) type 3 and two fibronectin type III (Fn3)-like domains. Expression plasmids coding for ChiB or deletion derivatives thereof were constructed in Escherichia coli. Deletion analysis showed that the ChtBD of ChiB plays an important role in efficient hydrolysis of insoluble chitin. The optimum pH and temperature of ChiB were 6.0 and 30°C, respectively. The enzyme showed relatively high catalysis, even at low temperatures close to 0°C, and remarkable thermal lability compared to ChiA and ChiC, which are the mesophilic chitinases of the same strain. The k cat /K m value for the ChiB reaction at 10°C was about 4.7 times higher than that of ChiC. These results suggest that ChiB is a cold-adapted enzyme. The RNA transcript of chiB was induced by 1% GlcNAc, and along with a rise in temperature, the RNA transcript showed a tendency to decrease. Thus, among the ChiA, ChiB, and ChiC chitinases, production of ChiB may be advantageous for the strain, allowing it to easily acquire nutrients from chitin and to survive in cold environments.Chitin, an insoluble homopolymer of ␤-(1,4)-linked N-acetylglucosamine (GlcNAc), is an abundant organic compound in nature. This polysaccharide is found in the exoskeletons and endoskeletons of many marine organisms, including mollusks, coelenterates, protozoa, fungi, and crustaceans. Since carbon and nitrogen are generally limited in the marine environment, chitin is a particularly important nutrient source for marine organisms. Chitinolytic marine bacteria play a crucial role in the recycling of chitinous materials for maintenance of the ecosystem in the marine environment (13). The concerted action of two chitinolytic enzymes, chitinase (EC 3.2.1.14) and ␤-N-acetylglucosaminidase (EC 3.2.1.30), is considered to be essential for the complete hydrolysis of chitin to GlcNAc. Chitinases cleave glycosidic linkages of GlcNAc randomly to produce soluble oligosaccharides, mainly chitobiose, which are further hydrolyzed to GlcNAc by ␤-N-acetylglucosaminidases. Finally, the degradation products, mainly GlcNAc, are then taken up by the cells as a carbon and nitrogen source.Alteromonas sp. strain O-7 is a gram-negative, flagellated, motile, and aerobic rod-shaped bacterium of marine origin and an efficient producer of chitinolytic enzymes (33). This strain produces at least three chitinases (ChiA, ChiB, and ChiC), a chitinase-like enzyme (ChiD), three ␤-N-acetylglucosaminidases (GlcNAcases A, B, and C), a transglycosylative enzyme (Hex99), a chitin-binding protein (Cbp1), and a chitin-binding protease (AprIV) in the presence of chitin. We have cloned and sequenced...

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The regulator CdsS/CdsR two-component system modulates expression of genes involved in chitin degradation of Pseudoalteromonas piscicida strain O-7

et al. 2007

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Pseudoalteromonas piscicida strain O-7 (formerly Alteromonas sp. strain O-7) is an efficient degrader of chitin in the marine environment. The chitinolytic system of the strain consists of many enzymes induced by N-acetylglucosamine (GlcNAc). This paper reports that CdsR, which is a response regulator of CdsS/CdsR two-component signal transduction system, is bound to near the promoter region of GlcNAc-induced aprIV gene. The CdsR protein as a response regulator was transphosphorylated by the CdsS protein as a sensor kinase. Furthermore, the transphosphorylation from CdsS to CdsR was promoted by chitin degradation products and a metabolite. The CdsR protein was also phosphorylated by acetyl phosphate which is an indicator of nutritive conditions of cells. Gel mobility shift assays demonstrated that phosphorylated CdsR (CdsR-P) was bound to not only near the promoter region of aprIV gene but also those of chiA, chiB, chiC, chiD and cbp1 genes which are induced in the presence of GlcNAc. Footprinting analysis demonstrated that CdsR-P was bound to the sequences around the transcriptional start sites of aprIV and chiD genes. These results indicate that CdsR is one of the common regulators of these genes involved in chitin degradation of the strain.

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Molecular Analysis of the Gene Encoding a Novel Chitin-Binding Protease from Alteromonas sp. Strain O-7 and Its Role in the Chitinolytic System

Cited by 27 publications

References 43 publications

Mechanistic Insight into the Function of the C-terminal PKD Domain of the Collagenolytic Serine Protease Deseasin MCP-01 from Deep Sea Pseudoalteromonas sp. SM9913

Mechanistic Insight into the Function of the C-terminal PKD Domain of the Collagenolytic Serine Protease Deseasin MCP-01 from Deep Sea Pseudoalteromonas sp. SM9913

Molecular Analysis of the Gene Encoding a Novel Cold-Adapted Chitinase (ChiB) from a Marine Bacterium, Alteromonas sp. Strain O-7

The regulator CdsS/CdsR two-component system modulates expression of genes involved in chitin degradation of Pseudoalteromonas piscicida strain O-7

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