Cloning and expression of a pectate lyase from the oral spirochete Treponema pectinovorum ATCC 33768.
The pelA gene, encoding a pectate lyase, from Treponema pectinovorum ATCC 33768 was isolated by heterologous expression of a cosmid library in Escherichia coli. In vitro transposon mutagenesis identified an open reading frame of 1293 bp capable of encoding a protein of 430 amino acids with a predicted amino-terminal signal sequence of 21 amino acids. Analysis of the amino acid sequence suggested that it is a member of the polysaccharide lyase family 10 of which all characterized members show pectate lyase activity. An amino-terminal His-tagged recombinant form of PelA was expressed and purified from E. coli. The recombinant enzyme has characteristics common to other bacterial pectate lyases such as an alkaline pH optimum, dependence on calcium ions for activity, and inhibition by zinc ions.
Walker
Stephen G
SG
Department of Oral Biology and Pathology, School of Dental Medicine, State University of New York, Stony Brook, NY 11794-8702, USA. stephen.walker@stonybrook.edu
Ryan
Maria E
ME
D000595:Amino Acid Sequence; D002122:Calcium Chloride / Q000494:pharmacology; D003001:Cloning, Molecular; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D005798:Genes, Bacterial; D016680:Genome, Bacterial; D015698:Genomic Library; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014169:Transformation, Bacterial; D014208:Treponema / Q000201:enzymology* / Q000235:genetics*; D019287:Zinc Sulfate / Q000494:pharmacology
D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D019287:Zinc Sulfate; D011133:Polysaccharide-Lyases; C023911:pectate lyase; D002122:Calcium Chloride