358-72-5Relevant academic research and scientific papers
Bacillus subtilis ypgA gene is fni, a nonessential gene encoding type 2 isopentenyl diphosphate isomerase
Takagi, Motoki,Kaneda, Kazuhide,Shimizu, Tomohiro,Hayakawa, Yoichi,Seto, Haruo,Kuzuyama, Tomohisa
, p. 132 - 137 (2004)
We previously identified the fni gene of Streptomyces sp. strain CL190 as type 2 isopentenyl diphosphate (IPP) isomerase, which needs both FMN and NADPH for enzyme activity. An fni gene homolog, ypgA, was detected in the database of the Bacillus subtilis genome. However, the ypgA product was about 140 amino acids shorter in the N-terminal than the Streptomyces fni gene product. A database search found three new putative start codons in 129, 225, and 411 bases upstream of the original start codon of the ypgA gene. The longest gene product, which was named ypgA3, showed the most significant homology to the Streptomyces fni gene product. The ypgA3 gene was expressed with an N-terminal His-tag in Escherichia coli and the purified soluble protein was characterized in detail. The ypgA3 protein converted IPP to its isomer dimethylallyl diphosphate in the presence of both FMN and NADPH. The enzyme also catalyzed the reverse reaction in the presence of both the cofactors. Disruption of the ypgA3 gene was not lethal to B. subtilis. These results indicate that Bacillus ypgA3 gene is fni, a nonessential gene encoding type 2 IPP isomerase.
Bioorganometallic mechanism of action, and inhibition, of IspH
Wang, Weixue,Wang, Ke,Liu, Yi-Liang,No, Joo-Hwan,Li, Jikun,Nilges, Mark J.,Oldfield, Eric
, p. 4522 - 4527 (2010)
We have investigated the mechanism of action of Aquifex aeolicus IspH [E-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase], together with its inhibition, using a combination of site-directed mutagenesis (K M;Vmax), EPR and 1H, 2H, 13C, 31P, and 57Fe-electron-nuclear double resonance (ENDOR) spectroscopy. On addition of HMBPP to an (unreactive) E126A IspH mutant, a reaction intermediate forms that has a very similar EPR spectrum to those seen previously with the HMBPP parent molecules, ethylene and allyl alcohol, bound to a nitrogenase FeMo cofactor. The EPR spectrum is broadened on 57Fe labeling and there is no evidence for the formation of allyl radicals. When combined with ENDOR spectroscopy, the results indicate formation of an organometallic species with HMBPP, a π?σ metallacycle or η2-alkenyl complex. The complex is poised to interact with H+ from E126 (and H124) in reduced wt IspH, resulting in loss of water and formation of an η1-allyl complex. After reduction, this forms an η3-allyl π-complex (i.e. containing an allyl anion) that on protonation (at C2 or C4) results in product formation. We find that alkyne diphosphates (such as propargyl diphosphate) are potent IspH inhibitors and likewise form metallacycle complexes, as evidenced by 1H, 2H, and 13C ENDOR, where hyperfine couplings of approximately 6 MHz for 13C and 10 MHz for 1H, are observed. Overall, the results are of broad general interest because they provide new insights into IspH catalysis and inhibition, involving organometallic species, and may be applicable to other Fe4S 4-containing proteins, such as IspG.
Isopentenyl diphosphate isomerase. Mechanism-based inhibition by diene analogues of isopentenyl diphosphate and dimethylallyl diphosphate
Wu, Zheng,Wouters, Johan,Poulter, C. Dale
, p. 17433 - 17438 (2005)
Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This is an essential step in the mevalonate entry into the isoprenoid biosynthetic pathway. The isomerization cat
Scalable Biosynthesis of the Seaweed Neurochemical, Kainic Acid
Chekan, Jonathan R.,McKinnie, Shaun M. K.,Moore, Malia L.,Poplawski, Shane G.,Michael, Todd P.,Moore, Bradley S.
, p. 8454 - 8457 (2019)
Kainic acid, the flagship member of the kainoid family of natural neurochemicals, is a widely used neuropharmacological agent that helped unravel the key role of ionotropic glutamate receptors, including the kainate receptor, in the central nervous system. Worldwide shortages of this seaweed natural product in the year 2000 prompted numerous chemical syntheses, including scalable preparations with as few as six-steps. Herein we report the discovery and characterization of the concise two-enzyme biosynthetic pathway to kainic acid from l-glutamic acid and dimethylallyl pyrophosphate in red macroalgae and show that the biosynthetic genes are co-clustered in genomes of Digenea simplex and Palmaria palmata. Moreover, we applied a key biosynthetic α-ketoglutarate-dependent dioxygenase enzyme in a biotransformation methodology to efficiently construct kainic acid on the gram scale. This study establishes both the feasibility of mining seaweed genomes for their biotechnological prowess.
Modular Chemoenzymatic Synthesis of Terpenes and their Analogues
Allemann, Rudolf K.,Benton, Jennifer C. R.,Dunbabin, Alice,Johnson, Luke A.,Mart, Robert J.
supporting information, p. 8486 - 8490 (2020/03/30)
Non-natural terpenoids offer potential as pharmaceuticals and agrochemicals. However, their chemical syntheses are often long, complex, and not easily amenable to large-scale production. Herein, we report a modular chemoenzymatic approach to synthesize terpene analogues from diphosphorylated precursors produced in quantitative yields. Through the addition of prenyl transferases, farnesyl diphosphates, (2E,6E)-FDP and (2Z,6Z)-FDP, were isolated in greater than 80 % yields. The synthesis of 14,15-dimethyl-FDP, 12-methyl-FDP, 12-hydroxy-FDP, homo-FDP, and 15-methyl-FDP was also achieved. These modified diphosphates were used with terpene synthases to produce the unnatural sesquiterpenoid semiochemicals (S)-14,15-dimethylgermacrene D and (S)-12-methylgermacrene D as well as dihydroartemisinic aldehyde. This approach is applicable to the synthesis of many non-natural terpenoids, offering a scalable route free from repeated chain extensions and capricious chemical phosphorylation reactions.
Probing the Substrate Promiscuity of Isopentenyl Phosphate Kinase as a Platform for Hemiterpene Analogue Production
Lund, Sean,Courtney, Taylor,Williams, Gavin J.
, p. 2217 - 2221 (2019/08/02)
Isoprenoids are a large class of natural products with wide-ranging applications. Synthetic biology approaches to the manufacture of isoprenoids and their new-to-nature derivatives are limited due to the provision in nature of just two hemiterpene buildin
Structure-based protein engineering enables prenyl donor switching of a fungal aromatic prenyltransferase
Mai, Peter,Zocher, Georg,Stehle, Thilo,Li, Shu-Ming
, p. 7461 - 7469 (2018/10/24)
Microorganisms provide valuable enzyme machinery to assemble complex molecules. Fungal prenyltransferases (PTs) typically catalyse highly regiospecific prenylation reactions that are of significant pharmaceutical interest. While the majority of PTs accepts dimethylallyl diphosphate (DMAPP), very few such enzymes can use geranyl diphosphate (GPP) or farnesyl diphosphate (FPP) as donors. This catalytic gap prohibits the wide application of PTs for structural diversification. Structure-guided molecular modelling and site-directed mutagenesis of FgaPT2 from Aspergillus fumigatus led to the identification of the gatekeeping residue Met328 responsible for the prenyl selectivity and sets the basis for creation of GPP- and FPP-accepting enzymes. Site-saturation mutagenesis of the gatekeeping residue at position 328 in FgaPT2 revealed that the size of this side chain is the determining factor for prenyl selectivity, while its hydrophobicity is crucial for allowing DMAPP and GPP to bind.
IspH-RPS1 and IspH-UbiA: rosetta stone proteins
Rao, Guodong,O'Dowd, Bing,Li, Jikun,Wang, Ke,Oldfield, Eric
, p. 6813 - 6822 (2015/11/24)
The protein IspH, (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate (HMPPP) reductase, is an essential 4Fe-4S cluster-containing protein in the methylerythritol phosphate pathway for isoprenoid biosynthesis. Using a sequence similarity network we found that there are >400 IspH proteins that are about twice as large as most of the IspHs studied to date since their IspH domains are fused to either the ribosomal protein S1 (RPS1), or to a UbiA (4-hydroxybenzoate octaprenyltransferase)-like protein. Many of the IspH-RPS1 proteins are present in anaerobes found in the human gut and some, such as Clostridium botulinum, C. tetani and Fusobacterium nucleatum, are pathogens. The IspH-UbiAs are all found in sulfate-reducing anaerobes. The IspH domains in IspH-RPS1 are fused to 4 and in a few cases 6 tandem repeats in RPS1 that, in most organisms, bind to mRNA or form part of the bacterial ribosome. Mutants in which the four RPS1 domains were sequentially eliminated had similar IspH activity as wild-type protein, indicating they are not essential for IspH catalysis. Overall, the results are of interest since they represent the first isolation of a catalytically active IspH-RPS1, as well as the identification of IspH-UbiA hybrids, two Rosetta stone proteins that are likely to be functionally related - IspH producing the isoprenoids required for a UbiA-like prenyltransferase; the IspH-RPS1 hybrids, perhaps, being involved in the stringent response or as Fe/O2 sensors.
Study of IspH, a key enzyme in the methylerythritol phosphate pathway using fluoro-substituted substrate analogues
Xiao, Youli,Chang, Wei-Chen,Liu, Hung-Wen,Liu, Pinghua
supporting information; experimental part, p. 5912 - 5915 (2011/12/16)
IspH, a [4Fe-4S]-cluster-containing enzyme, catalyzes the reductive dehydroxylation of 4-hydroxy-3-methyl-butenyl diphosphate (HMBPP) to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the methylerythritol phosphate pathway. Studies
Linear free energy relationships demonstrate a catalytic role for the flavin mononucleotide coenzyme of the type II isopentenyl diphosphate: Dimethylallyl diphosphate isomerase
Thibodeaux, Christopher J.,Chang, Wei-Chen,Liu, Hung-Wen
supporting information; experimental part, p. 9994 - 9996 (2010/10/04)
The type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) catalyzes the reversible isomerization of the two ubiquitous isoprene units, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are required to initiate the biosynthesis of all isoprenoid compounds found in nature. The overall chemical transformation catalyzed by IDI-2 involves a net 1,3-proton addition/elimination reaction. Surprisingly, IDI-2 requires a reduced flavin mononucleotide (FMN) coenzyme to carry out this redox neutral isomerization. The exact function of FMN in catalysis has not yet been clearly defined. To provide mechanistic insight into the role of the reduced flavin in IDI-2 catalysis, several FMN analogues with altered electronic properties were chemoenzymatically prepared, and their effects on the kinetic properties of the IDI-2 catalyzed reaction were investigated. Linear free energy relationships (LFERs) between the electronic properties of the flavin and the steady state kinetic parameters of the IDI-2 catalyzed reaction were observed. The LFER studies are complemented with kinetic isotope effect studies and kinetic characterization of an active site mutant enzyme (Q154N). Cumulatively, the data presented in this work (and in other studies) suggest that the reduced FMN coenzyme of IDI-2 functions as an acid/base catalyst, with the N5 atom of the flavin likely playing a critical role in the deprotonation of IPP en route to DMAPP formation. Several potential chemical mechanisms involving the reduced flavin as an acid/base catalyst are presented and discussed.
