Recombinant anaerobic maize aldolase: overexpression, characterization, and metabolic implications.
Complementary DNA sequence of anaerobically induced cytoplasmic maize aldolase was expressed under control of the tac promoter sequence in Escherichia coli using the pKK223-3 plasmid as a vehicle. Levels of recombinant protein expressed exceeded 20 mg of soluble aldolase per liter of culture. The purified recombinant enzyme displayed the expected molecular weight and tetrameric subunit assembly on the basis of mobilities on denaturing electrophoretic gels and gel filtration, respectively. Sequencing of the NH2 terminus and amino acid composition analysis of the recombinant protein including COOH-terminal peptides agreed with the cDNA sequence. Partial kinetic characterization based on product inhibition studies was consistent with the ordered uni-bi reaction mechanism expected of aldolases. Turnover with respect to substrates Fru-1,6-P2 and Fru-1-P by the recombinant enzyme is the highest reported to date for class I aldolases. Fru-1,6-P2 cleavage rate by recombinant cytoplasmic maize enzyme is three times greater than that of the chloroplast enzyme. Fru-1-P cleavage is 8-fold greater than that of the rabbit liver isozyme and 20-fold greater than that of the rabbit muscle isozyme to which maize aldolase exhibits the greatest homology. The implications of such a high Fru-1-P turnover on carbohydrate utilization under anaerobiosis is discussed.
Berthiaume
L
L
Département de biochimie, Faculté de médecine, Université de Sherbrooke, Quebec, Canada.
Beaudry
D
D
Lazure
C
C
Tolan
D R
DR
Sygusch
J
J
D000595:Amino Acid Sequence; D000693:Anaerobiosis; D004099:Dihydroxyacetone Phosphate / Q000494:pharmacology; D005634:Fructose-Bisphosphate Aldolase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D004099:Dihydroxyacetone Phosphate; D005634:Fructose-Bisphosphate Aldolase