2.5.1.82: hexaprenyl diphosphate synthase [geranylgeranyl-diphosphate specific]
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Reaction
Synonyms
(all-E) hexaprenyl diphosphate synthase, (all-trans) hexaprenyl diphosphate synthase, EC 2.5.1.33, hexaprenyl pyrophosphate synthase, HexPPs, HexPS
ECTree
2.5.1.82 hexaprenyl diphosphate synthase [geranylgeranyl-diphosphate specific]Advanced search results
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Engineering on EC 2.5.1.82 - hexaprenyl diphosphate synthase [geranylgeranyl-diphosphate specific]
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PROTEIN VARIANTS 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
additional information
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introduction of mutations mimicking the chain-length determination region of short-chain enzymes into Sulfolobus solfataricus hexaprenyldiphosphate synthase and characterization of the mutated enzymes. To mimic short-chain enzymes without geranylgeranyl diphosphate synthase type III, the typical amino acid residues observed in the region of enzymes of each group are introduced into Sulfolobus solfataricus hexaprenyldiphosphate synthase. Several successive amino acids before the first aspartate rich motif of Sulfolobus solfataricus hexaprenyldiphosphate synthase are replaced with those of geranylgeranyl diphosphate synthase from Saccharomyces cerevisiae to construct a mutant mimicking geranylgeranyl diphosphate synthase type III. To facilitate purification, (His)6-tag is introduced at the N-terminal of each enzyme. Introduction of the tag does not significantly affect the activity and product specificity of wild-type Sulfolobus solfataricus hexaprenyldiphosphate synthase. The mutant HPS-F1, HPS-GG1, and HPS-GG3, mimicking the chain-length determination regions of eukaryotic farnesyl diphosphate synthases, archaeal geranylgeranyl diphosphate synthases, and eukaryotic geranylgeranyl diphosphate synthases, respectively, sustain high activity comparable to that of the wild type enzyme, except for the facts that HPS-F1 shows reduced activity when (all-trans)-geranylgeranyl diphosphate is used as the allylic substrate (5.5% of the activity of wild-type HPS) and that the activity of HPS-GG3 toward geranyl diphosphate is relatively low (16% of that of wild type hexaprenyldiphosphate synthase). HPS-GG2, possessing the insertion sequence to mimic the chain-length determination region of geranylgeranyl diphosphate synthase type II, significantly decreases enzyme activity toward each allylic substrate (0.8%, 8.3%, and 45% of the activities of wild-type HPS for geranyl diphosphate, farnesyl diphosphate, and (all-trans)-geranylgeranyl diphosphate, respectively). HPS-F2 mimicking the chain-length determination region of farnesyl diphosphate synthase type II, which has both the insertion and the additional mutation before first aspartate rich motif, shows no activity, although the same amounts of enzymes are used. The inactivation of HPS-F2 is considered to arise from either the difference of its insertion sequence from that of HPS-GG2 or the introduction of bulky amino acid before first aspartate rich motif. When geranyldiphosphate and farnesyl diphosphate are used as the allylic substrate, the main product of HPS-F1 is geranylgeranyl diphosphate. A slight amount of C25 product is produced when (all-trans)-geranylgeranyl diphosphate is the substrate. These results and the low reactivity of HPS-F1 with (all-trans)-geranylgeranyl diphosphate indicate that the product specificity of the mutant has been changed into that of geranylgeranyl diphosphate synthase. The conversion of Sulfolobus solfataricus hexaprenyldiphosphate synthase into geranylgeranyl diphosphate synthase, not into farnesyl diphosphate synthase, by the mutation mimicking the chain-length determination region of farnesyl diphosphate synthase type I might reflect the structural difference between hexaprenyldiphosphate synthase and type I farnesyl diphosphate synthases existing at positions other than the mutated sites. HPS-GG1 maily produces hexaprenyldiphosphate, just as wild-type hexaprenyl diphosphate synthase does. It also yields small amounts of C35 product and, when geranyl diphosphate is used as the substrate, the C40 product. Either the conformation of the chain-length determination region or the orientation of the carbon-chain of products in the cavity of the enzyme is considerably different between the archaeal geranylgeranyl diphosphate synthase and hexaprenyl diphosphate synthase
