Genetic and biochemical characterization of an exopolygalacturonase and a pectate lyase from Yersinia enterocolitica.
Yersinia enterocolitica, an invasive foodborne human pathogen, degrades polypectate by producing two depolymerizing enzymes, pectate lyase (PL) and polygalacturonase (PG). The gene encoding the PG activity, designated pehY, was located in a 3-kb genomic fragment of Y. enterocolitica ATCC 49397. The complete nucleotide sequence of this 3-kb fragment was determined and an open reading frame consisting of 1803 bp was predicted to encode a PG protein with an estimated M(r) of 66 kDa and pI of 6.3. The amino acid sequence of prePG showed 59 and 43% identity to that of the exopolygalacturonase (exoPG) of Erwinia chrysanthemi and Ralstonia solanacearum, respectively. The Y. enterocolitica PG overproduced in Escherichia coli was purified to near homogeneity using perfusion cation exchange chromatography. Analysis of the PG depolymerization products by high performance anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD) revealed the exolytic nature of this enzyme. The Y. enterocolitica PL overproduced in E. coli was also partially purified and the M(r) and pI were estimated to be 55 kDa and 5.2, respectively. HPAEC-PAD analysis of the PL depolymerization products indicated the endolytic nature of this enzyme. Southern hybridization analyses revealed that pehY and pel genes of Y. enterocolitica are possibly encoded in the chromosome rather than in the plasmid. Purified exopolygalacturonase (over 10 activity units) was unable to macerate plant tissues.
Liao
C H
CH
Eastern Regional Research Center, U.S. Department of Agriculture, Wyndmoor, PA 19038, USA. cliao@arserrc.gov
Revear
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L
Hotchkiss
A
A
Savary
B
B
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000379:methods; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel; D016680:Genome, Bacterial; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D015008:Yersinia enterocolitica / Q000201:enzymology*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C020136:exopolygalacturonase; D011133:Polysaccharide-Lyases; C023911:pectate lyase