Pyridoxal 5'-phosphate dependent histidine decarboxylase: overproduction, purification, biosynthesis of soluble site-directed mutant proteins, and replacement of conserved residues.
The hdc gene coding for the pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii has been expressed in Escherichia coli under control of the lac promoter. The enzyme accumulates to 7-8% of total cell protein and is purified to homogeneity by passage through three columns. Fourteen site-directed mutant enzymes were constructed to explore the roles of residues of interest, especially those in the sequence Ser229-X230-His231-N epsilon-(phosphopyridoxylidene)Lys232, since identical sequences also appear in several other decarboxylases. Most of the overproduced mutant proteins were aggregated into inclusion bodies, but when the late log phase cultures were cooled from 37 to 25 degrees C before induction, the mutant proteins were obtained as soluble products. Ala or Cys in place of Ser-229 yielded mutant enzymes about 7% as active as wild-type, indicating that this serine residue is not essential for catalysis but contributes to activity through conformational or other effects. Of the replacements made for His-231 (Asn, Gln, Phe, and Arg), only Gln and Asn gave partially active enzymes (about 12% and 0.2% of wild-type, respectively). The side-chain amide of Gln may act by mimicking the positionally equivalent tau-nitrogen on the imidazole ring of histidine to provide an interaction (e.g., a hydrogen bond) required for efficient catalysis. The Lys-232 residue that interacts with pyridoxal 5'-phosphate appears central to catalytic efficiency since replacing it with Ala yields a mutant protein that is virtually inactive but retains the ability to bind both pyridoxal 5'-phosphate and histidine efficiently.(ABSTRACT TRUNCATED AT 250 WORDS)
Vaaler
G L
GL
Department of Microbiology, University of Texas, Austin 78712.
Snell
E E
EE
D000595:Amino Acid Sequence; D002262:Carboxy-Lyases / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics; D006640:Histidine Decarboxylase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D007763:Lac Operon; D009154:Mutation; D010957:Plasmids; D011511:Proteus / Q000201:enzymology*; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D012995:Solubility; D013329:Structure-Activity Relationship; D013696:Temperature
D011732:Pyridoxal Phosphate; D004247:DNA; D002262:Carboxy-Lyases; D006640:Histidine Decarboxylase