The Preparation of Glucosamine Oligosaccharides. I. Separation<sup>1,2</sup>

Horowitz, Sylvia T.; Roseman, Saul; Blumenthal, Harold J.

doi:10.1021/ja01575a059

Cited by 157 publications

(42 citation statements)

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“…The following chemicals, reagents, and materials were purchased from the indicated sources. p-nitrophenyl ␤-D-Nacetylglucosaminide (PNP-GlcNAc) was from Sigma; Hepes buffer was from Fisher; oligonucleotide primers were from IDT (Coralville, IA); chitin oligosaccharides (GlcNAc)n, n ϭ 2-6, were prepared as described (15) or were from Seikagaku (Rockville, MD); DNA restriction and modifying enzymes were from New England Biolabs; and pGEM-T Vector System I was from Promega. Other buffers and reagents were of the highest purity commercially available.…”

Section: Methodsmentioning

confidence: 99%

The chitinolytic cascade in Vibrios is regulated by chitin oligosaccharides and a two-component chitin catabolic sensor/kinase

Roseman

2003

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

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Chitin, a highly insoluble polymer of GlcNAc, is produced in massive quantities in the marine environment. Fortunately for survival of aquatic ecosystems, chitin is rapidly catabolized by marine bacteria. Here we describe a bacterial two-component hybrid sensor͞ kinase (of the ArcB type) that rigorously controls expression of Ϸ50 genes, many involved in chitin degradation. The sensor gene, chiS, was identified in Vibrio furnissii and Vibrio cholerae (predicted amino acid sequences, full-length: 84% identical, 93% similar). Mutants of chiS grew normally on GlcNAc but did not express extracellular chitinase, a specific chitoporin, or ␤-hexosaminidases, nor did they exhibit chemotaxis, transport, or growth on chitin oligosaccharides such as (GlcNAc) 2. Expression of these systems requires three components: wild-type chiS; a periplasmic highaffinity chitin oligosaccharide, (GlcNAc) n (n > 1), binding protein (CBP); and the environmental signal, (GlcNAc) n. Our data are consistent with the following model. In the uninduced state, CBP binds to the periplasmic domain of ChiS and ''locks'' it into the minus conformation. The environmental signal, (GlcNAc) n, dissociates the complex by binding to CBP, releasing ChiS, yielding the plus phenotype (expression of chitinolytic genes). In V. cholerae, a cluster of 10 contiguous genes (VC0620 -VC0611) apparently comprise a (GlcNAc) 2 catabolic operon. CBP is encoded by the first, VC0620, whereas VC0619 -VC0616 encode a (GlcNAc) 2 ABC-type permease. Regulation of chiS requires expression of CBP but not (GlcNAc) 2 transport. (GlcNAc)n is suggested to be essential for signaling these cells that chitin is in the microenvironment.

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Section: Methodsmentioning

confidence: 99%

The chitinolytic cascade in Vibrios is regulated by chitin oligosaccharides and a two-component chitin catabolic sensor/kinase

Roseman

2003

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

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“…Whatman GF/F glass microfiber filters, and thin layer chromatography plates (Silica-Gel 60) were purchased from EM Science (Cherry Hill, NJ). (GlcNAc) 2 was prepared as described (11) or by modifications to be described elsewhere. Other buffers and reagents were of the highest purity commercially available.…”

Section: Methodsmentioning

confidence: 99%

The Chitin Disaccharide,N,N′-Diacetylchitobiose, Is Catabolized byEscherichia coli and Is Transported/Phosphorylated by the Phosphoenolpyruvate:Glycose Phosphotransferase System

Keyhani¹,

Wang²,

Lee

et al. 2000

Journal of Biological Chemistry

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3 H]Me-TCB uptake system was induced only by (GlcNAc) n , n ‫؍‬ 2 or 3. The apparent K m of transport was 50 -100 M, and effective inhibitors of uptake included (GlcNAc) n , n ‫؍‬ 2 or 3, cellobiose, and other PTS sugars, i.e. glucose and GlcNAc. Presumably the PTS sugars inhibit by competing for PTS components. Kinetic properties of the transport system are described.We have reported that wild type Escherichia coli and a mutant unable to transport GlcNAc can utilize the chitin disaccharide, N,NЈ-diacetylchitobiose or (GlcNAc) 2 1 as the sole source of carbon for growth (6).2 An E. coli transposon mutant was selected that was unable to grow on (GlcNAc) 2 but behaved normally on GlcNAc, and the mutant was used to clone the (GlcNAc) 2 catabolic operon. Sequence analysis of the genes in the operon compared with the sequence of the complete E. coli genome (7) showed that the (GlcNAc) 2 operon corresponded to the previously described "cryptic" cellobiose operon (8, 9) In the preliminary report (6) we demonstrated that the wild type allele of this operon encoded for (GlcNAc) 2 but not cellobiose utilization; the cel genes were therefore renamed and constitute the chb (N,NЈ-diacetylchito-biose) operon. In earlier work (10), a (GlcNAc) 2 permease was described in the Gram-negative, chitinolytic marine bacterium Vibrio furnissii. For these experiments, we employed a nonhydrolyzable (GlcNAc) 2 analogue, methyl ␤-N,NЈ- H]Me-TCB). In V. furnissii, the transport product was characterized as unmodified [3 H]Me-TCB, and its apparent K m for uptake was Ͻ1 M.In the present studies, we show that transport of (GlcNAc) 2 / [ 3 H]Me-TCB in E. coli is mediated via the phosphoenolpyruvate:glycose phosphotransferase system (PTS), with the sugar accumulated as its phosphorylated derivative. The phosphoryl group is linked to the C-6 position of the nonreducing GlcNAc. In E. coli, the apparent K m for [ 3 H]Me-TCB uptake is 50 -100 M. Thus, the chitin disaccharide, (GlcNAc) 2 , is transported by different mechanisms in these two closely related organisms. This is in sharp contrast to other sugars, as discussed below. EXPERIMENTAL PROCEDURESMaterials-Reagents for bacterial media were obtained from Difco Laboratories (Detroit, MI), J. T. Baker (Phillipsburg, NJ), and BBL Microbiology Systems (Cockeysville, MD). Molecular biology reagents were obtained from New England Biolabs (Beverly, MA), U.S. Biochemical Corp. (Cleveland, OH), and Stratagene (La Jolla, CA). Radioisotopes were purchased from Life Science Products. Whatman GF/F glass microfiber filters, and thin layer chromatography plates (Silica-Gel 60) were purchased from EM Science (Cherry Hill, NJ). (GlcNAc) 2 was prepared as described (11) or by modifications to be described elsewhere. Other buffers and reagents were of the highest purity commercially available. PTS proteins, such as homogeneous Enzyme I, HPr, and IIA Glc , were kind gifts from Drs. Norman Meadow, Regina Savtchenko, and Roshan Mattoo.Bacterial Strains-E. coli strain XL1-Blue MR (D(mcrA)183 ⌬(mcrCB-hsdSMR-mrr)173 endA...

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“…Commercial chitosan was completely deacetylated by fusion with KOH (23), and the chitosan reacetylated with 3 H-labeled acetic anhydride (24,25). In the latter step, the solution is vigorously stirred, and as the chitin is formed it yields very fine particles, close to a colloidal dispersion that makes a good substrate for chitinases (25).…”

Section: Methodsmentioning

confidence: 99%

The Chitin Catabolic Cascade in the Marine Bacterium Vibrio furnissii

Keyhani¹,

Roseman

1996

Journal of Biological Chemistry

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Chitin catabolism in Vibrio furnissii comprises several signal transducing systems and many proteins. Two of these enzymes are periplasmic and convert chitin oligosaccharides to GlcNAc and (GlcNAc) 2 . One of these unique enzymes, a chitodextrinase, designated EndoI, is described here.The protein, isolated from a recombinant Escherichia coli clone, exhibited (via SDS-polyacrylamide gel electrophoresis) two enzymatically active, close running bands (ϳmass of 120 kDa) with identical N-terminal sequences. The chitodextrinase rapidly cleaved chitin oligosaccharides, (GlcNAc) 4 to (GlcNAc) 2 , and (GlcNAc) 5,6 to (GlcNAc) 2 and (GlcNAc) 3 . EndoI was substrate inhibited in the millimolar range and was inactive with chitin, glucosamine oligosaccharides, glycoproteins, and glycopeptides containing (GlcNAc) 2 .The sequence of the cloned gene indicates that it encodes a 112,690-kDa protein (1046 amino acids). Both proteins lacked the predicted N-terminal 31 amino acids, corresponding to a consensus prokaryotic signal peptide. Thus, E. coli recognizes and processes this V. furnissii signal sequence. Although inactive with chitin, the predicted amino acid sequence of EndoI displayed similarities to many chitinases, with 8 amino acids completely conserved in 10 or more of the homologous proteins. There was, however, no "consensus" chitin-binding domain in EndoI.The marine bacterium Vibrio furnissii can utilize chitin as a sole source of carbon and nitrogen; the cascade involves several signal transduction processes and a multitude of proteins (1-4) whose expression is stringently regulated. These cells also rapidly catabolize GlcNAc and soluble chitin oligosaccharides, and the rate of GlcNAc utilization is comparable to that for the optimal rate of glucose catabolism by Escherichia coli.The enzymatic hydrolysis of chitin has been studied for almost a century (5). In 1939, Zechmeister and Toth (6, 7) reported the key observation; the insoluble polymer was converted to its monomer, GlcNAc, by two enzymes, a "polysaccharidase" (chitinase), and a "disaccharidase," a ␤-Nacetylglucosaminidase or chitobiase. Chitinases and chitobiases have since been purified from a wide variety of sources (8) including plants (9, 10), animals (11), yeast (12), and prokaryotes such as Streptomyces (13, 14), Bacillus circulans (15, 16), and Vibrio parahaemolyticus (17). In addition, recent reports describe similar enzymes in higher animals. Although their exact specificities and functions remain to be determined, enzymes capable of cleaving chitin oligosaccharides have been reported in human plasma (18), and in articular chondrocytes and synovial cells (19). Chitotriosidase activity is elevated more than 600-fold in the plasma of Gaucher patients (18).As noted in the references and reviews cited above, many of the structural genes encoding these enzymes have been cloned and sequenced. In agreement with the early work, the chitinases yield (GlcNAc) 2 as the major product of chitin hydrolysis, although higher oligomers are sometimes intermediates in ...

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The Preparation of Glucosamine Oligosaccharides. I. Separation^1,2

Cited by 157 publications

References 1 publication

The chitinolytic cascade in Vibrios is regulated by chitin oligosaccharides and a two-component chitin catabolic sensor/kinase

The chitinolytic cascade in Vibrios is regulated by chitin oligosaccharides and a two-component chitin catabolic sensor/kinase

The Chitin Disaccharide,N,N′-Diacetylchitobiose, Is Catabolized byEscherichia coli and Is Transported/Phosphorylated by the Phosphoenolpyruvate:Glycose Phosphotransferase System

The Chitin Catabolic Cascade in the Marine Bacterium Vibrio furnissii

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The Preparation of Glucosamine Oligosaccharides. I. Separation1,2

Cited by 157 publications

References 1 publication

The chitinolytic cascade in Vibrios is regulated by chitin oligosaccharides and a two-component chitin catabolic sensor/kinase

The chitinolytic cascade in Vibrios is regulated by chitin oligosaccharides and a two-component chitin catabolic sensor/kinase

The Chitin Disaccharide,N,N′-Diacetylchitobiose, Is Catabolized byEscherichia coli and Is Transported/Phosphorylated by the Phosphoenolpyruvate:Glycose Phosphotransferase System

The Chitin Catabolic Cascade in the Marine Bacterium Vibrio furnissii

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The Preparation of Glucosamine Oligosaccharides. I. Separation^1,2