Chaperonin-mediated assembly of wild-type and mutant subunits of human propionyl-CoA carboxylase expressed in Escherichia coli.
We developed a bacterial expression system for the human alpha and beta cDNAs of propionyl-CoA carboxylase (PCC). These cDNAs (less the putative mitochondrial matrix targeting presequences) were co-expressed in Escherichia coli on one plasmid vector with each cDNA having its own IPTG-inducible promoter. Only negligible amounts of active PCC were measured despite the presence of both alpha and beta subunits as indicated by Western blot analysis and the almost complete biotinylation of the alpha subunit. Co-expression of this plasmid with a second plasmid vector over-expressing the E. coli chaperonin proteins, groES and groEL, resulted in a several hundred-fold increase in PCC specific activity, to a level comparable with that found in crude human liver extracts. PCC was partially purified on monomeric avidin affinity resin and the presence of both alpha and beta subunits was demonstrated, thereby confirming the assembly of both subunits into an active enzyme. Deficiency of either alpha PCC or beta PCC results in propionic acidemia, an autosomal recessive disorder. We used this expression system to characterize one missense mutation previously described in five Japanese alleles, namely C1283T (Thr428lle) in beta PCC. This mutation, when expressed in E.coli under the same conditions as that of wild-type PCC, had null activity, despite the presence of assembled alpha PCC and beta PCC subunits. This bacterial expression system can be useful for analysis of either alpha PCC or beta PCC mutations. Our findings indicated that the groES and groEL chaperonin proteins were essential for folding and assembly of the human PCC heteromeric subunits.
Kelson
T L
TL
Department of Pediatrics, University of Colorado School of Medicine, Denver 80262, USA.
Ohura
T
T
Kraus
J P
JP
D015153:Blotting, Western; D002262:Carboxy-Lyases / Q000096:biosynthesis* / Q000235:genetics*; D018835:Chaperonin 10 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D018833:Chaperonins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D043586:Methylmalonyl-CoA Decarboxylase; D009154:Mutation; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
D018835:Chaperonin 10; D011994:Recombinant Proteins; D018833:Chaperonins; D002262:Carboxy-Lyases; D043586:Methylmalonyl-CoA Decarboxylase