Direct Evidence of an Enzyme-Generated LPP Intermediate in (+)-Limonene Synthase Using a Fluorinated GPP Substrate Analog

Morehouse, B.R.; Kumar, Rajeev; Matos, Jason O.; Yu, Qi; Bannister, Austin M.; Malik, Karan; Temme, J. Sebastian; Krauss, Isaac J.; Oprian, Daniel D.

doi:10.1021/acschembio.9b00514

Cited by 11 publications

(20 citation statements)

References 35 publications

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“…The crystals were then soaked with the non-reactive substrate analog 12,13-difluorofarnesyl diphosphate (DFFPP), which we prepared by enzymatic elongation of 8,9-difluorogeranyl diphosphate (DFGPP) 21,22 with isopentenyl diphosphate (IPP; Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

“…To explore the mechanism underlying the initial anti-Markovnikov cyclization in the PS reaction, we first prepared a nonreactive substrate analogue, 12,13-difluorofarnesyl diphosphate (DFFPP), by enzymatic elongation of 8,9-difluorogeranyl diphosphate (DFGPP) , with isopentenyl diphosphate [IPP (Figure )]. The two electronegative fluorine atoms in DFFPP are positioned to inhibit the reactivity of the π-electrons in the C10C11 bond and thus block the initial cyclization reaction.…”

Section: Resultsmentioning

confidence: 99%

“…, and E,E-FPP caryolan-1-ol HO as previously described for substrates and substrate analogs of the enzyme (+)-limonene synthase. 21,46,47 The purity of the diphosphate products was assessed by proton, carbon, and phosphorus NMR. NMR spectra were recorded on a Varian 400-MR spectrometer (9.4 T, 400…”

Section: Discussionmentioning

confidence: 99%

“…The synthesis and purification of DMAPP, IPP, and E,E -FPP from the corresponding allylic alcohols (Sigma-Aldrich, St. Louis, MO) was performed as previously described for substrates and substrate analogues of the enzyme (+)-limonene synthase. ,, The purity of the diphosphate products was assessed by proton, carbon, and phosphorus nuclear magnetic resonance (NMR). NMR spectra were recorded on a Varian 400-MR spectrometer (9.4 T, 400 MHz) in D 2 O adjusted to pD ∼8.4 with ND 4 OD.…”

Section: Methodsmentioning

confidence: 99%

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Mechanism Underlying Anti-Markovnikov Addition in the Reaction of Pentalenene Synthase

et al. 2020

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Most terpene synthase reactions follow Markovnikov rules for formation of high energy carbenium ion intermediates. However, there are notable exceptions. For example, pentalenene synthase (PS) undergoes an initial anti-Markovnikov cyclization reaction followed by a 1,2hydride shift to form an intermediate humulyl cation with positive charge on the secondary carbon C9 of the farnesyl diphosphate substrate. The mechanism by which these enzymes stabilize and guide regioselectivity of secondary carbocations has not heretofore been elucidated.In an effort to better understand these reactions, we grew crystals of apo-PS, soaked them with the non-reactive substrate analog 12,13-difluorofarnesyl diphosphate, and solved the x-ray structure of the resulting complex at 2.2 Å resolution. The most striking feature of the active site structure is that C9 is positioned 3.5 Å above the center of the side chain benzene ring of residue F76, perfectly poised for stabilization of the charge through a cation-p interaction. In addition, the main chain carbonyl of I177 and neighboring intramolecular C6,C7-double bond are positioned to stabilize the carbocation by interaction with the face opposite that of F76.Mutagenesis experiments also support a role for residue 76 in cation-p interactions. Most interesting is the F76W mutant which gives a mixture of products that likely result from stabilizing a positive charge on the adjacent secondary carbon C10 in addition to C9 as in the wild-type enzyme. The crystal structure of the F76W mutant clearly shows carbons C9 and C10 centered above the fused benzene and pyrrole rings of the indole side chain, respectively, such that a carbocation at either position could be stabilized in this complex, and two anti-Markovnikov products, pentalenene and humulene, are formed. Finally, we show that there is a rough correlation (although not absolute) of an aromatic side chain (F or Y) at position 76 in related terpene synthases from Streptomyces that catalyze similar anti-Markovnikov addition reactions.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Mechanism Underlying Anti-Markovnikov Addition in the Reaction of Pentalenene Synthase

et al. 2020

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“…caryolan-1-ol HO as previously described for substrates and substrate analogs of the enzyme (+)-limonene synthase. 21,46,47 Technologies (New Jersey). The genes were in a pET-28a (+) vector as a NcoI (5'-end) and…”

Section: Discussionmentioning

confidence: 99%

Mechanism Underlying Anti-Markovnikov Addition in the Reaction of Pentalenene Synthase

Matos

Kumar

Alison

et al. 2020

Preprint

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Accession CodesThe atomic coordinates and structure factors for the DFFPP-PS complex have been deposited in the Protein Data Bank. RCSB PDB entry 6WKC (WT-Mg), 6WKD (WT-DFFPP), 6WKE DFFPP) for the respective models. ABBREVIATIONS FPP, farnesyl diphosphate; DFFPP, 12,13-difluorofarnesyl diphosphate; PS, pentalenene synthase, CS, caryolan-1-ol synthase. SUPPORTING INFORMATION AVAILABLE AbstractMost terpene synthase reactions follow Markovnikov rules for formation of high energy carbenium ion intermediates. However, there are notable exceptions. For example, pentalenene synthase (PS) undergoes an initial anti-Markovnikov cyclization reaction followed by a 1,2hydride shift to form an intermediate humulyl cation with positive charge on the secondary carbon C9 of the farnesyl diphosphate substrate. The mechanism by which these enzymes stabilize and guide regioselectivity of secondary carbocations has not heretofore been elucidated.In an effort to better understand these reactions, we grew crystals of apo-PS, soaked them with the non-reactive substrate analog 12,13-difluorofarnesyl diphosphate, and solved the x-ray structure of the resulting complex at 2.2 Å resolution. The most striking feature of the active site structure is that C9 is positioned 3.5 Å above the center of the side chain benzene ring of residue F76, perfectly poised for stabilization of the charge through a cation-p interaction. In addition, the main chain carbonyl of I177 and neighboring intramolecular C6,C7-double bond are positioned to stabilize the carbocation by interaction with the face opposite that of F76.Mutagenesis experiments also support a role for residue 76 in cation-p interactions. Most interesting is the F76W mutant which gives a mixture of products that likely result from stabilizing a positive charge on the adjacent secondary carbon C10 in addition to C9 as in the wild-type enzyme. The crystal structure of the F76W mutant clearly shows carbons C9 and C10 centered above the fused benzene and pyrrole rings of the indole side chain, respectively, such that a carbocation at either position could be stabilized in this complex, and two anti-Markovnikov products, pentalenene and humulene, are formed. Finally, we show that there is a rough correlation (although not absolute) of an aromatic side chain (F or Y) at position 76 in related terpene synthases from Streptomyces that catalyze similar anti-Markovnikov addition reactions.

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Synthetic biology, combinatorial biosynthesis, and chemo‑enzymatic synthesis of isoprenoids

Malico

Calzini

Gayen

et al. 2020

Journal of Industrial Microbiology and Biotechnology

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Isoprenoids are a large class of natural products with myriad applications as bioactive and commercial compounds. Their diverse structures are derived from the biosynthetic assembly and tailoring of their scaffolds, ultimately constructed from two C5 hemiterpene building blocks. The modular logic of these platforms can be harnessed to improve titers of valuable isoprenoids in diverse hosts and to produce new-to-nature compounds. Often, this process is facilitated by the substrate or product promiscuity of the component enzymes, which can be leveraged to produce novel isoprenoids. To complement rational enhancements and even re-programming of isoprenoid biosynthesis, high-throughput approaches that rely on searching through large enzymatic libraries are being developed. This review summarizes recent advances and strategies related to isoprenoid synthetic biology, combinatorial biosynthesis, and chemo-enzymatic synthesis, focusing on the past 5 years. Emerging applications of cell-free biosynthesis and high-throughput tools are included that culminate in a discussion of the future outlook and perspective of isoprenoid biosynthetic engineering.

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Direct Evidence of an Enzyme-Generated LPP Intermediate in (+)-Limonene Synthase Using a Fluorinated GPP Substrate Analog

Cited by 11 publications

References 35 publications

Mechanism Underlying Anti-Markovnikov Addition in the Reaction of Pentalenene Synthase

Mechanism Underlying Anti-Markovnikov Addition in the Reaction of Pentalenene Synthase

Mechanism Underlying Anti-Markovnikov Addition in the Reaction of Pentalenene Synthase

Synthetic biology, combinatorial biosynthesis, and chemo‑enzymatic synthesis of isoprenoids

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