Isolation and Expression in
 Escherichia coli
 of
 cslA
 and
 cslB
 , Genes Coding for the Chondroitin Sulfate-Degrading Enzymes Chondroitinase AC and Chondroitinase B, Respectively, from
 Flavobacterium heparinum

Tkalec, A. L.; Fink, D; Blain, F.; Zhang-Sun, G.; Laliberte, M.; Bennett, Dorothy C.; Gu, Kenan; Zimmermann, Joe; Su, Hong Myung

doi:10.1128/aem.66.1.29-35.2000

Cited by 54 publications

(34 citation statements)

References 44 publications

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“…However, the lower lytic activity observed in the native periplasmic extract may be possibly explained by the presence of low percentage of ChonAC in native extract, and it may also be true that ChonAC concentration is low in proteins of the bacteria at least in cultured conditions, as little ChonAC activity was observed in non-induced condition. Tkalec et al (2000) also detected lytic activity of recombinant chondroitin sulphate-degrading enzyme of F. heparinum expressed in E. coli with the expression vector pIBXI. Stringer-Roth et al (2002), by using a more accurate metric method, detected activities of extracellular ChonAC in non-induced culture supernatants in different strains of F. columnare, but the bacterium was cultured in different medium with peptone being the major component.…”

Section: Characteristics Of F Columnare Gene Csla Encoding Chonacmentioning

confidence: 99%

Gene cloning and functional analysis of glycosaminoglycan-degrading enzyme chondroitin AC lyase from Flavobacterium columnare G4

et al. 2005

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The chondroitin AC lyase gene, cslA, was cloned for the first time from the fish bacterial pathogen F. columnare G 4 . From the first transcription initiation site, the cslA extends 2620 nucleotides to the end of the 3¢ region. The open reading frame of cslA transcript has 2286 nucleotides encoding 762 amino acids with a 16 residues long signal peptide at the Nterminus. The gene, cslA was then successfully expressed in Escherichia coli and recombinant chondroitin AC lyase, rChonAC was purified, with its lytic activity analyzed. Zymography analysis copolymerized with chondroitin sulphate revealed the lytic activity of rChonAC and also the crude native ChonAC isolated from periplamic space of cultured F. columnare G 4 . The low level of lytic activity observed in crude native ChonAC may be due possibly to the low level of expression of this gene in the cultured condition. The expression and the role of this virulence factor is of interest for further research on the pathogenesis of F. columnare.

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Section: Characteristics Of F Columnare Gene Csla Encoding Chonacmentioning

confidence: 99%

Gene cloning and functional analysis of glycosaminoglycan-degrading enzyme chondroitin AC lyase from Flavobacterium columnare G4

et al. 2005

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“…The soluble protein fraction was isolated by centrifugation (10,000 Â g, 4 jC, 10 min) and checked for lyase AC activity with chondroitin-4-sulfate as substrate. Expression of mutant lyase AC protein was confirmed by sodium dodecyl sulfate (SDS)-PAGE and Western blotting using polyclonal anti-lyase AC antibodies as previously described [27].…”

Section: Methodsmentioning

confidence: 99%

“…Gradient polyacrylamide gel electrophoresis (PAGE) analysis was performed on a 20 cm vertical slab gel (ProteankII) from Bio-Rad, Richmond, CA. The F. heparinum AC lyase gene with upstream Hep1 promoter was cloned as previously described [27]. For sitedirected mutagenesis, the oligonucleotide primers used to introduce the Arg292Ala mutation had sequences 5V -TAGAAGGCCGCGGAGTAAGTGCACCAGACATTC-TAAATAAAAAGGC-3Vand 5V -GCCTTTTTATTTA-GAATGTCTGGTGCACTTACTCCGCGGCCTTCTA-3V for the forward and reverse primers, respectively.…”

Section: Methodsmentioning

confidence: 99%

Role of arginine 292 in the catalytic activity of chondroitin AC lyase from Flavobacterium heparinum

Capila

Rethwisch

et al. 2002

Biochimica et Biophysica Acta (BBA) - Protein Structure and Mol

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Chondroitin AC lyase (chondroitinase EC 4.2.2.5), an eliminase from Flavobacterium heparinum, cleaves chondroitin sulfate glycosaminoglycans (GAGs) at 1,4 glycosidic linkages between N-acetylgalactosamine and glucuronic acid residues. Cleavage occurs through h-elimination in a random endolytic action pattern. Crystal structures of chondroitin AC lyase (wild type) complexed with oligosaccharides reveal a binding site within a narrow and shallow protein channel, suggesting several amino acids as candidates for the active site residues. Site-specific mutagenesis studies on residues within the active-site tunnel revealed that only the Arg to Ala 292 mutation (R292A) retained activity. Furthermore, structural data suggested that R292 was primarily involved in recognition of N-acetyl or O-sulfo moieties of galactosamine residues and did not directly participate in catalysis. The current study demonstrates that the R292A mutation affords f 10-fold higher K m values but no significant change in V max , consistent with hypothesis that R292 is involved in binding the O-sulfo moiety of the saccharide residues. Change in chondroitin sulfate viscosity, as a function of its enzymatic cleavage, affords a shallower concave curve for the R292A mutant, suggesting its action pattern is neither purely random endolytic nor purely random exolytic. Product studies using gel electrophoresis confirm the altered action pattern of this mutant. Thus, these data suggest that the R292A mutation effectively reduces binding affinity, making it possible for the oligosaccharide chain, still bound after initial endolytic cleavage, to slide through the tunnel to the catalytic site for subsequent, processive, step-wise, exolytic cleavage. D

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“…Few genes from F. heparinum have been studied at the molecular level. DNA sequences are available only for the five glycosaminoglycan lyase genes hepA, B, C and cslA, B (Sasisekharan et al, 1993 ;Su et al, 1996 ;Tkalec et al, 2000). Among them, it appeared that hepA was expressed at the highest level when grown in heparinonly medium (Lohse & Linhardt, 1992).…”

Section: Construction Of a Conjugation/integration Plasmid For F Hepmentioning

confidence: 99%

Development of a genetic system for the transfer of DNA into Flavobacterium heparinum The GenBank accession number for the sequence reported in this paper is AF221716.

Su¹,

Shao²,

Tkalec³

et al. 2001

Microbiology

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Flavobacterium heparinum (now Pedobacter heparinus) is a Gram-negative soil bacterium which can produce yellow pigments. It synthesizes five enzymes that degrade glycosoaminoglycan molecules. The study of this unique bacterium has been limited by the absence of a genetic manipulation system. In this paper, the construction of a conjugation/integration plasmid system and a broad-host-range plasmid, both of which contain a F. heparinum functional selective marker created by placing the trimethoprim resistance gene, dhfrII, under the control of the hepA regulatory region is described. Both plasmids were introduced into F. heparinum by conjugation and/or electroporation, and trimethoprim resistant colonies were obtained. Fifty electroporants were obtained per microgram covalently closed circular plasmid DNA. The existence of integrated plasmid DNA was confirmed by Southern hybridization and PCR. The existence of a derivative of the broad-host-range plasmid pBBR1 in F. heparinum was demonstrated by plasmid digestion and Southern hybridization, and by transformation of Escherichia coli.

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Isolation and Expression in Escherichia coli of cslA and cslB , Genes Coding for the Chondroitin Sulfate-Degrading Enzymes Chondroitinase AC and Chondroitinase B, Respectively, from Flavobacterium heparinum

Cited by 54 publications

References 44 publications

Gene cloning and functional analysis of glycosaminoglycan-degrading enzyme chondroitin AC lyase from Flavobacterium columnare G4

Gene cloning and functional analysis of glycosaminoglycan-degrading enzyme chondroitin AC lyase from Flavobacterium columnare G4

Role of arginine 292 in the catalytic activity of chondroitin AC lyase from Flavobacterium heparinum

Development of a genetic system for the transfer of DNA into Flavobacterium heparinum The GenBank accession number for the sequence reported in this paper is AF221716.

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