Introduction of a new branchpoint in tetrapyrrole biosynthesis in Escherichia coli by co-expression of genes encoding the chlorophyll-specific enzymes magnesium chelatase and magnesium protoporphyrin methyltransferase.
The genes encoding the three Mg chelatase subunits, ChlH, ChlI and ChlD, from the cyanobacterium Synechocystis PCC6803 were all cloned in the same pET9a-based Escherichia coli expression plasmid, forming an artificial chlH-I-D operon under the control of the strong T7 promoter. When a soluble extract from IPTG-induced E. coli cells containing the pET9a-ChlHID plasmid was assayed for Mg chelatase activity in vitro, a high activity was obtained, suggesting that all three subunits are present in a soluble and active form. The chlM gene of Synechocystis PCC6803 was also cloned in a pET-based E. coli expression vector. Soluble extract from an E. coli strain expressing chlM converted Mg-protoporphyrin IX to Mg-protoporphyrin monomethyl ester, demonstrating that chlM encodes the Mg-protoporphyrin methyltransferase of Synechocystis. Co-expression of the chlM gene together with the chlH-I-D construct yielded soluble protein extracts which converted protoporphyrin IX to Mg-protoporphyrin IX monomethyl ester without detectable accumulation of the Mg-protoporphyrin IX intermediate. Thus, active Mg chelatase and Mg-protoporphyrin IX methyltransferase can be coupled in E. coli extracts. Purified ChlI, -D and -H subunits in combination with purified ChlM protein were subsequently used to demonstrate in vitro that a molar ratio of ChlM to ChlH of 1 to 1 results in conversion of protoporphyrin IX to Mg-protoporphyrin monomethyl ester without significant accumulation of Mg-protoporphyrin.
Jensen
P E
PE
Department of Plant Biology, The Royal Veterinary and Agricultural University, Frederiksberg C, Denmark.
Gibson
L C
LC
Shephard
F
F
Smith
V
V
Hunter
C N
CN
D002734:Chlorophyll / Q000096:biosynthesis; D000458:Cyanobacteria / Q000201:enzymology / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D005818:Genetic Engineering; D008190:Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009876:Operon; D011758:Pyrroles / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D045725:Tetrapyrroles
D011758:Pyrroles; D011994:Recombinant Proteins; D045725:Tetrapyrroles; D002734:Chlorophyll; D008780:Methyltransferases; C060234:magnesium-protoporphyrin methyltransferase; D008190:Lyases; C086788:magnesium chelatase