title: 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.
abstract: 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.
authors.affiliation: Department of Plant Biology, The Royal Veterinary and Agricultural University, Frederiksberg C, Denmark.
authors.forename: P E
authors.initials: PE
authors.lastname: Jensen
authors.forename: L C
authors.initials: LC
authors.lastname: Gibson
authors.forename: F
authors.initials: F
authors.lastname: Shephard
authors.forename: V
authors.initials: V
authors.lastname: Smith
authors.forename: C N
authors.initials: CN
authors.lastname: Hunter
mesh_terms: 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
chemical_list: D011758:Pyrroles; D011994:Recombinant Proteins; D045725:Tetrapyrroles; D002734:Chlorophyll; D008780:Methyltransferases; C060234:magnesium-protoporphyrin methyltransferase; D008190:Lyases; C086788:magnesium chelatase