title: Exceptionally high percentage of IPP synthesis by Ginkgo biloba IspH is mainly due to Phe residue in the active site.
abstract: (E)-4-Hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) reductase (IspH, HDR or LytB) is an Fe/S enzyme catalyzing the reductive dehydroxylation of HMBPP to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the last step of methylerythritol phosphate (MEP) pathway. The MEP pathway is known to produce 4-6:1 ratio of IPP and DMAPP mixture by the last enzyme, IspH. Plant IspH in plastids follows same catalytic mechanism as others, but GbIspH (Ginkgo biloba IspH) was reported to produce a mixture of IPP and DMAPP in a ratio of 16:1. Present catalytic mechanisms of IspH involve a common allyl anion intermediate, and the intramolecular proton transfer to the allyl moiety is considered as the key reaction step determining the product between IPP and DMAPP. The F212 residue in plant IspH was found as a potential amino acid residue that could mediate the proton transfer to the allyl anion intermediate before the product release. In this report, catalytic function of GbIspH F212 residue (H74 in E. coli), especially during the product formation in the active site, was studied by means of site-directed mutation. The product ratio of IPP/DMAPP was measured as 6.5 ± 0.1 for F212H GbIspH, and the value was close to the reported bacterial IspH having His residue on that specific position. Along with the other F212Y mutant, of which ratio was determined as 10.9 ± 0.1, the results strongly support that the Phe residue in plant IspH is the key amino acid residue that allows exclusive production of IPP in plant chloroplast.
authors.affiliation: Metalloenzyme Research Group and Department of Integrative Plant Science, Chung-Ang University, Anseong, 456-756, South Korea.
authors.forename: Bok-Kyu
authors.initials: BK
authors.lastname: Shin
authors.affiliation: Metalloenzyme Research Group and Department of Integrative Plant Science, Chung-Ang University, Anseong, 456-756, South Korea.
authors.forename: Mihyang
authors.initials: M
authors.lastname: Kim
authors.affiliation: Metalloenzyme Research Group and Department of Integrative Plant Science, Chung-Ang University, Anseong, 456-756, South Korea. Electronic address: jaehongh@cau.ac.kr.
authors.forename: Jaehong
authors.initials: J
authors.lastname: Han
mesh_terms: D020134:Catalytic Domain; D002736:Chloroplasts / Q000378:metabolism; D020441:Ginkgo biloba / Q000737:chemistry* / Q000378:metabolism; D045782:Hemiterpenes / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010755:Organophosphates / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism*; D011487:Protein Conformation; D013237:Stereoisomerism; D013440:Sulfides / Q000378:metabolism
chemical_list: C467061:4-hydroxy-3-methyl-2-butenyl diphosphate; D045782:Hemiterpenes; D010755:Organophosphates; D009943:Organophosphorus Compounds; D013440:Sulfides; C011342:pyrite; C004809:isopentenyl pyrophosphate; C043060:3,3-dimethylallyl pyrophosphate; D007501:Iron
keywords: Dimethylallyl diphosphate; Fe/S protein; Isopentenyl diphosphate; IspH; Methylerythritol phosphate; Regioselectivity