Purification, crystallization and properties of porphobilinogen deaminase from a recombinant strain of Escherichia coli K12.
Porphobilinogen deaminase has been purified and crystallized from an overproducing recombinant strain of Escherichia coli harbouring a hemC-containing plasmid which has permitted the purification of milligram quantities of the enzyme. Determination of the Mr of the enzyme by SDS/polyacrylamide-gel electrophoresis (35,000) and gel filtration (32,000) agrees with the gene-derived Mr of 33,857. The enzyme has a Km of 19 +/- 7 microM, an isoelectric point of 4.5 and an N-terminal sequence NH2-MLDNVLRIAT. The substrate, porphobilinogen, binds to the active-site dipyrromethane cofactor to form three intermediate complexes: ES, ES2 and ES3. The gene-derived primary structure of the E. coli deaminase is compared with that derived from the cDNA of the human enzyme.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000642:Ammonia-Lyases / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005818:Genetic Engineering; D006863:Hydrogen-Ion Concentration; D011163:Hydroxymethylbilane Synthase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011162:Porphobilinogen / Q000378:metabolism
D000596:Amino Acids; D046911:Macromolecular Substances; D011162:Porphobilinogen; D011163:Hydroxymethylbilane Synthase; D000642:Ammonia-Lyases