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

2020 ◽  
Author(s):  
Jason O. Matos ◽  
Ramasamy P. Kumar ◽  
Alison C. Ma ◽  
MacKenzie Patterson ◽  
Isaac J. Krauss ◽  
...  
Keyword(s):  
Positive Charge ◽  
High Energy ◽  
Terpene Synthase ◽  
Side Chain ◽  
Aromatic Side Chain ◽  
Terpene Synthases ◽  
In The Wild ◽  
Pentalenene Synthase ◽  
Markovnikov Addition ◽  
Secondary Carbon

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 1,2-hydride 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-π 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-π 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.

Effects of environment on flavin reactivity in morphinone reductase: analysis of enzymes displaying differential charge near the N-1 atom and C-2 carbonyl region of the active-site flavin

2001 ◽  
Vol 359 (2) ◽  
pp. 315-323 ◽  
Author(s):  
Daniel H. CRAIG ◽  
Terez BARNA ◽  
Peter C. E. MOODY ◽  
Neil C. BRUCE ◽  
Stephen K. CHAPMAN ◽  
...  
Keyword(s):  
Active Site ◽  
Positive Charge ◽  
Hydride Transfer ◽  
Reduced Form ◽  
Side Chain ◽  
Wild Type ◽  
Reduction Potentials ◽  
In The Wild ◽  
Mutant Forms ◽  
Positively Charged

The side chain of residue Arg238 in morphinone reductase (MR) is located close to the N-1/C-2 carbonyl region of the flavin isoalloxazine ring. During enzyme reduction negative charge develops in this region of the flavin. The positioning of a positively charged side chain in the N-1/C-2 carbonyl region of protein-bound flavin is common to many flavoprotein enzymes. To assess the contribution made by Arg238 in stabilizing the reduced flavin in MR we isolated three mutant forms of the enzyme in which the position of the positively charged side chain was retracted from the N-1/C-2 carbonyl region (Arg238 → Lys), the positive charge was removed (Arg238 → Met) or the charge was reversed (Arg238 → Glu). Each mutant enzyme retains flavin in its active site. Potentiometric studies of the flavin in the wild-type and mutant forms of MR indicate that the flavin semiquinone is not populated to any appreciable extent. Reduction of the flavin in each enzyme is best described by a single Nernst function, and the values of the midpoint reduction potentials (E12) for each enzyme fall within the region of −247±10mV. Stopped-flow studies of NADH binding to wild-type and mutant MR enzymes reveal differences in the kinetics of formation and decay of an enzyme–NADH charge-transfer complex, reflecting small perturbations in active-site geometry. Reduced rates of hydride transfer in the mutant enzymes are attributed to altered geometrical alignment of the nicotinamide coenzyme with FMN rather than major perturbations in reduction potential, and this is supported by an observed entropy–enthalpy compensation effect on the hydride transfer reaction throughout the series of enzymes. The data indicate, in contrast with dogma, that the presence of a positively charged side chain close to the N-1/C-2 carbonyl region of the flavin in MR is not required to stabilize the reduced flavin. This finding may have general implications for flavoenzyme catalysis, since it has generally been assumed that positive charge in this region has a stabilizing effect on the reduced form of flavin.

Modeling the Effects of O-sulfonation on the CID of Serine

2020 ◽  
Author(s):  
Kenneth Lucas ◽  
George Barnes
Keyword(s):  
Sulfo Group ◽  
High Energy ◽  
Empirical Method ◽  
Side Chain ◽  
Energy Structure ◽  
Direct Dynamics ◽  
Theoretical Mass ◽  
Intermolecular Proton Transfer ◽  
Dynamics Simulations ◽  
Semi Empirical

We present the results of direct dynamics simulations and DFT calculations aimed at elucidating the effect of \textit{O}-sulfonation on the collision induced dissociation for serine. Towards this end, direct dynamics simulations of both serine and sulfoserine were performed at multiple collision energies and theoretical mass spectra obtained. Comparisons to experimental results are favorable for both systems. Peaks related to the sulfo group are identified and the reaction dynamics explored. In particular, three significant peaks (m\z 106, 88, and 81) seen in the theoretical mass spectrum directly related to the sulfo group are analyzed as well as major peaks shared by both systems. Our analysis shows that the m\z 106 peaks result from intramolecular rearrangements, intermolecular proton transfer among complexes composed of initial fragmentation products, and at high energy side-chain fragmentation. The \mz 88 peak was found to contain multiple constitutional isomers, including a previously unconsidered, low energy structure. It was also seen that the RM1 semi empirical method was not able to obtain all of the major peaks seen in experiment for sulfoserine. In contrast, PM6 did obtain all major experimental peaks.

Synthesis of Substituted -N-(5-((7-Methyl-2-Oxo-2H-Chromen-4-yl)-Methyl)-1,3,4-Thiadiazol-2-yl)-Benzamide Derivatives Using TBTU As Coupling Agent And Their Evaluation For Anti Tubercular Activity

2021 ◽  
Vol 18 ◽  
Author(s):  
Monika Kakadiya ◽  
Yunus Pasha ◽  
Malleshappa Noolvi ◽  
Ashish Patel
Keyword(s):  
Antitubercular Activity ◽  
Side Chain ◽  
Antitubercular Agents ◽  
Facile Synthesis ◽  
Aromatic Side Chain ◽  
Reaction Conditions ◽  
Coupling Reagent ◽  
H37rv Strain ◽  
Benzamide Derivatives

: Tuberculosis remains a highly infectious disease across the world. In the identification of new antitubercular agents, coumarin clubbed thiadiazole amides have been synthesized and evaluated for in vitro antitubercular activity. Due to the growing concern about chemicals and their impact on the environment, greener and faster reaction conditions needed to be incorporated. Therefore, we used TBTU as a coupling reagent for efficient and facile synthesis of substituted-N-(5-((7-methyl-2-oxo-2H-chromes-4-yl)-methyl)-1,3, 4 - thiadiazol-2-yl)-benzamide 4a-j with good yields up to 95% in mild reaction condition. All the synthesized compounds were evaluated in vitro for antitubercular activity against the H37Rv strain of M.Tuberculosis. Compounds 4c, 4f, and 4j were found active at 25 µg/mL against M. tb H37Rv. Electron withdrawing substituents present on aromatic side-chain showed promising anti-tubercular activity.

scholarly journals Transcriptome and proteome of the corm, leaf and flower of Hypoxis hemerocallidea (African potato)

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0253741
Author(s):  
Mihai-Silviu Tomescu ◽  
Selisha Ann Sooklal ◽  
Thuto Ntsowe ◽  
Previn Naicker ◽  
Barbara Darnhofer ◽  
...  
Keyword(s):  
De Novo ◽  
Differential Expression Analysis ◽  
Terpene Synthase ◽  
Future Research ◽  
Terpene Synthases ◽  
Illumina Hiseq ◽  
Prostate Hyperplasia ◽  
Inflammatory Conditions ◽  
Urinary Infections ◽  
Hypoxis Hemerocallidea

The corm of Hypoxis hemerocallidea, commonly known as the African potato, is used in traditional medicine to treat several medical conditions such as urinary infections, benign prostate hyperplasia, inflammatory conditions and testicular tumours. The metabolites contributing to the medicinal properties of H. hemerocallidea have been identified in several studies and, more recently, the active terpenoids of the plant were profiled. However, the biosynthetic pathways and the enzymes involved in the production of the terpene metabolites in H. hemerocallidea have not been characterised at a transcriptomic or proteomic level. In this study, total RNA extracted from the corm, leaf and flower tissues of H. hemerocallidea was sequenced on the Illumina HiSeq 2500 platform. A total of 143,549 transcripts were assembled de novo using Trinity and 107,131 transcripts were functionally annotated using the nr, GO, COG, KEGG and SWISS-PROT databases. Additionally, the proteome of the three tissues were sequenced using LC-MS/MS, revealing aspects of secondary metabolism and serving as data validation for the transcriptome. Functional annotation led to the identification of numerous terpene synthases such as nerolidol synthase, germacrene D synthase, and cycloartenol synthase amongst others. Annotations also revealed a transcript encoding the terpene synthase phytoalexin momilactone A synthase. Differential expression analysis using edgeR identified 946 transcripts differentially expressed between the three tissues and revealed that the leaf upregulates linalool synthase compared to the corm and the flower tissues. The transcriptome as well as the proteome of Hypoxis hemerocallidea presented here provide a foundation for future research.

scholarly journals Identification of Sesquiterpene Synthases from Nostoc punctiforme PCC 73102 and Nostoc sp. Strain PCC 7120

2008 ◽  
Vol 190 (18) ◽  
pp. 6084-6096 ◽  
Author(s):  
Sean A. Agger ◽  
Fernando Lopez-Gallego ◽  
Thomas R. Hoye ◽  
Claudia Schmidt-Dannert
Keyword(s):  
Functional Expression ◽  
Terpene Synthase ◽  
Putative Hybrid ◽  
Nostoc Punctiforme ◽  
Terpene Synthases ◽  
E Coli ◽  
Reductase Gene ◽  
Pcc 7120 ◽  
Defense Compound

ABSTRACT Cyanobacteria are a rich source of natural products and are known to produce terpenoids. These bacteria are the major source of the musty-smelling terpenes geosmin and 2-methylisoborneol, which are found in many natural water supplies; however, no terpene synthases have been characterized from these organisms to date. Here, we describe the characterization of three sesquiterpene synthases identified in Nostoc sp. strain PCC 7120 (terpene synthase NS1) and Nostoc punctiforme PCC 73102 (terpene synthases NP1 and NP2). The second terpene synthase in N. punctiforme (NP2) is homologous to fusion-type sesquiterpene synthases from Streptomyces spp. shown to produce geosmin via an intermediate germacradienol. The enzymes were functionally expressed in Escherichia coli, and their terpene products were structurally identified as germacrene A (from NS1), the eudesmadiene 8a-epi-α-selinene (from NP1), and germacradienol (from NP2). The product of NP1, 8a-epi-α-selinene, so far has been isolated only from termites, in which it functions as a defense compound. Terpene synthases NP1 and NS1 are part of an apparent minicluster that includes a P450 and a putative hybrid two-component protein located downstream of the terpene synthases. Coexpression of P450 genes with their adjacent located terpene synthase genes in E. coli demonstrates that the P450 from Nostoc sp. can be functionally expressed in E. coli when coexpressed with a ferredoxin gene and a ferredoxin reductase gene from Nostoc and that the enzyme oxygenates the NS1 terpene product germacrene A. This represents to the best of our knowledge the first example of functional expression of a cyanobacterial P450 in E. coli.

Synthesis of new cyclopeptide analogues of the miuraenamides

2021 ◽  
Vol 18 ◽  
Author(s):  
Sarah Kappler ◽  
Andreas Siebert ◽  
Uli Kazmaier
Keyword(s):  
Natural Products ◽  
Biological Properties ◽  
Amide Bond ◽  
Side Chain ◽  
Aromatic Side Chain ◽  
Highly Active ◽  
C Terminus ◽  
Terminal Amino ◽  
High Cytotoxicity ◽  
Derivatives Of

Introduction: Miuraenamides belong to marine natural compounds with interesting biological properties. Materials and Methods: They initiate polymerization of monomeric actin and therefore show high cytotoxicity by influencing the cytoskeleton. New derivatives of the miuraenamides have been synthesized containing a N-methylated amide bond instead of the more easily hydrolysable ester in the natural products. Results: Incorporation of an aromatic side chain onto the C-terminal amino acid of the tripeptide fragment also led to highly active new miuraenamides. Conclusion: We could show that the ester bond of the natural product miuraenamide can be replaced by an N-methyl amide. The yields in the cyclization step are high and generally much better that with the corresponding esters. On the other hand, the biological activity of the new amide analogs are lower compared to the natural products, but the activity can significantly be increased by incorporation of a p-nitrophenyl group at the C-terminus of the peptide fragment.

Transforming growth factor alpha: an aromatic side chain at position 38 is essential for biological activity

1989 ◽  
Vol 9 (2) ◽  
pp. 860-864
Author(s):  
E Lazar ◽  
E Vicenzi ◽  
E Van Obberghen-Schilling ◽  
B Wolff ◽  
S Dalton ◽  
...  
Keyword(s):  
Biological Activity ◽  
Growth Factor ◽  
Disulfide Bonds ◽  
Transforming Growth Factor ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Transforming Growth Factor Alpha ◽  
Aromatic Side Chain ◽  
Factor Alpha ◽  
Alpha Gene

Site-directed mutagenesis has been performed in the human transforming growth factor alpha gene. When tyrosine 38 is mutated into phenylalanine or tryptophane, biological activity is retained. In contrast, other alterations between cysteine 34 and cysteine 43 and disruption of disulfide bonds 8 to 21 and 34 to 43 resulted in loss of activities. The presence of an aromatic side chain at position 38 of transforming growth factor alpha seems to be essential for its activity.

Sign in / Sign up

Export Citation Format

Share Document