Heterologous expression of cDNAs encoding Chlorella sorokiniana NADP-specific glutamate dehydrogenase wild-type and mutant subunits in Escherichia coli cells and comparison of kinetic and thermal stability properties of their homohexamers.
Full-length cDNAs encoding the alpha- and beta-subunits and a truncated mutant subunit of the Chlorella sorokiniana NADP-GDH isozymes were constructed and expressed in Escherichia coli cells. The kinetic and thermal stability properties of the resultant homohexamers were examined. The electrophoretic mobility of the recombinant alpha- and beta-subunits was identical to that of the native subunits as determined by immunoblotting. The homohexamers were purified by anion-exchange and gel-filtration chromatography. The alpha- and beta-homohexamers that were synthesized in the bacterial cells were shown to have similar Michaelis constants for their substrates as previously shown after synthesis in C. sorokiniana cells (Bascomb and Schmidt, 1987). The alpha homohexamer synthesized in the bacterium was allosteric with respect to NADPH but to a lesser degree than when isolated from the alga. The mutant homohexamer was composed of subunits that were truncated by 40 amino acids at their N-termini. This mutant isozyme was kinetically similar to the larger, anabolic alpha-homohexamer, but it did not display the allosteric response to NADPH shown by the alpha-homohexamer. The three isozymes had significant thermal tolerance and were stable at 50 degrees C. The temperature optimum for catalytic activity for the alpha- and beta-homohexamers was 60 degrees C, and 65 degrees C for the delta40N homohexamer. This study demonstrated that most of the kinetic properties of the Chlorella sorokiniana NADP-GDH isozymes were retained after their synthesis in a heterologous system, and that the distinctive N-terminal domains of these isozymes have dramatic effects on their biochemical characteristics.
Lawit
Shai J
SJ
Microbiology and Cell Science Department, University of Florida, PO Box 110700, Building 981, Gainesville, FL 32611-0700, USA.
Miller
Philip W
PW
Dunn
Waltraud I
WI
Mirabile
Jeremy S
JS
Schmidt
Robert R
RR
D000595:Amino Acid Sequence; D002708:Chlorella / Q000201:enzymology*; D018076:DNA, Complementary / Q000235:genetics*; D019281:Dimerization; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D018086:Glutamate Dehydrogenase (NADP+) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D018076:DNA, Complementary; D021122:Protein Subunits; D011994:Recombinant Proteins; D018086:Glutamate Dehydrogenase (NADP+)