Co-overexpression of bacterial GroESL chaperonins partly overcomes non-productive folding and tetramer assembly of E. coli-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) carrying the prevalent disease-causing K304E mutation.
The influence of co-overexpression of the bacterial chaperonins GroEL and GroES on solubility, tetramer formation and enzyme activity of three variants of heterologously-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) was analysed in order to investigate the molecular mechanism underlying MCAD deficiency caused by the prevalent K304E mutation. Depending on which of the three amino acids--lysine (wild-type), glutamic acid (K304E) or glutamine (K304Q) are present at position 304 of the mature polypeptide, three different patterns were observed in our assay system: (i) solubility, tetramer formation and yield of enzyme activity of wild-type MCAD is largely independent of GroESL co-overexpression; (ii) the larger part of the K304Q mutant is insoluble without and solubility is enhanced with GroESL co-overexpression; solubility correlates with the amount of tetramer detected and the enzyme activity measured as observed for the wild-type protein. (iii) Solubility of the K304E mutant is in a similar fashion GroESL responsive as the K304Q mutant, but the amount of tetramer observed and the enzyme activity measured do not correlate with the amount of soluble K304E MCAD protein detected in Western blotting. In a first attempt to estimate the specific activity, we show that tetrameric K304E and K304Q mutant MCAD display a specific activity in the range of the wild-type enzyme. Taken together, our results strongly suggest, that the K304E mutation primarily impairs the rate of folding and subunit assembly. Based on the data presented, we propose that lysine-304 is important for the folding pathway and that an exchange of this amino acid both to glutamine or glutamic acid leads to an increased tendency to misfold/aggregate. Furthermore, exchange of lysine-304 with an amino acid with negative charge at position 304 (glutamic acid) but not with a neutral charge (glutamine) negatively affects conversion to active tetramers. A possible explanation for this latter effect--charge repulsion upon subunit docking--is discussed.
Bross
P
P
Dept. of Clinical Chemistry, Arhus Kommunehospital and Skejby Sygehus, Arhus, Denmark.
Andresen
B S
BS
Winter
V
V
Kräutle
F
F
Jensen
T G
TG
Nandy
A
A
Kølvraa
S
S
Ghisla
S
S
Bolund
L
L
Gregersen
N
N
D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D018833:Chaperonins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005971:Glutamates; D018698:Glutamic Acid; D005973:Glutamine; D006801:Humans; D008239:Lysine; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D017510:Protein Folding; D011506:Proteins / Q000378:metabolism*
D001426:Bacterial Proteins; D005971:Glutamates; C097970:GroESL protein, Bacteria; D011506:Proteins; D005973:Glutamine; D018698:Glutamic Acid; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase; D018833:Chaperonins; D008239:Lysine