Lidia Smentek

Quantitative exploration of the catalytic landscape separating divergent plant sesquiterpene synthases.

Throughout molecular evolution, organisms create assorted chemicals in response to varying ecological niches. Catalytic landscapes underlie metabolic evolution, wherein mutational steps alter the biosynthetic properties of enzymes. We report the first systematic quantitative characterization of a catalytic landscape underlying the evolution of sesquiterpene chemical diversity. Based on our previous discovery of a set of 9 naturally occurring amino acid substitutions that functionally inter-converted orthologous sesquiterpene synthases from Nicotiana tabaccum and Hyoscyamus muticus, we created a library of all possible residue combinations (29 = 512) in the N. tabaccum parent. The product spectra of 418 active enzymes to reveal a rugged landscape where several minimal combinations of the 9 mutations encode convergent solutions to the inter-conversions of parental activities. Quantitative comparisons indicate context dependence for mutational effects - epistasis - in product specificity and promiscuity. These results provide a measure of the mutational accessibility of phenotypic variability among a diverging lineage of terpene synthases.

Structural Elucidation of Cisoid and Transoid Cyclization Pathways of a Sesquiterpene Synthase using 2-Fluorofarnesyl Diphosphates

Sesquiterpene skeletal complexity in nature originates from the enzyme-catalyzed ionization of (trans,trans)-farnesyl diphosphate (FPP) (1a) and subsequent cyclization along either 2,3-transoid or 2,3-cisoid farnesyl cation pathways. Tobacco 5-epi-aristolochene synthase (TEAS), a transoid synthase, produces cisoid products as a component of its minor product spectrum. To investigate the cryptic cisoid cyclization pathway in TEAS, we employed (cis,trans)-FPP (1b) as an alternative substrate. Strikingly, TEAS was catalytically robust in the enzymatic conversion of (cis,trans)-FPP (1b) to exclusively (≥99.5%) cisoid products. Further, crystallographic characterization of wild-type TEAS and a catalytically promiscuous mutant (M4 TEAS) with 2-fluoro analogues of both all-trans FPP (1a) and (cis,trans)-FPP (1b) revealed binding modes consistent with preorganization of the farnesyl chain. These results provide a structural glimpse into both cisoid and transoid cyclization pathways efficiently templated by a single enzyme active site, consistent with the recently elucidated stereochemistry of the cisoid products. Further, computational studies using density functional theory calculations reveal concerted, highly asynchronous cyclization pathways leading to the major cisoid cyclization products. The implications of these discoveries for expanded sesquiterpene diversity in nature are discussed.

Concerted, highly asynchronous, enzyme-catalyzed [4 + 2] cycloaddition in the biosynthesis of spinosyn A; computational evidence

A theoretical study has been carried out on model systems to study a recently reported, (Nature, 2011, 473, 109) biosynthetic, [4 + 2] cycloaddition catalyzed by a stand-alone enzyme (the cyclase SpnF). It was suggested in this paper that SpnF is the first known example of a Diels-Alderase (DA). In the present study, for a model system of the substrate a transition structure was found with density functional calculations (DFT). In addition, the intrinsic reaction coordinate calculations indicated that the transition structure is that of a concerted, but highly asynchronous, DA reaction. Based on the DFT and Møller-Plesset second order calculations the activation energy was estimated to be about 15 kcal mol(-1). The results of a natural population analysis indicated that there is significant charge transfer in the transition state, and it is proposed that possibly the enzyme plays a dual role of not only folding the substrate into the proper conformation for the DA reaction to occur, but also lowering its activation energy by stabilization of the highly polarized transition structure.

Compelling computational evidence for the concerted cyclization of the ABC rings of hopene from protonated squalene.

The long-standing question of what is the nature of the cyclization of squalene to form tetracyclic and pentacyclic triterpenes has been addressed computationally. Using the DFT method with an intrinsic reaction coordinate calculation, we find that the first three rings of protonated squalene were formed without the intermediacy of mono- or bicyclic carbocations. The cyclization, calculated in the gas phase, proceeds in a highly asynchronous, concerted reaction to yield the tricyclic, tertiary carbocation with a 5-membered C ring. The fourth double bond of squalene is not properly oriented for the ring expansion of the C ring in concert with the formation of the 5-membered ring.

Theoretical description of the spectroscopic properties of rare earth ions in crystals

Abstract In this article the present state of knowledge of the theory of one- and two-photon processes observed in rare earth ions in crystals is presented. The conclusions are based on the results of ab initio calculations performed for various ions across the lanthanide series. The model applied for the calculations is based on the Rayleigh–Schrodinger perturbation theory, and the amplitude of a certain electric dipole transition is expressed in terms of effective operators. The radial integrals of the effective operators are defined by the perturbed functions that contain the perturbing influence of single excitations from the 4f shell to all one-electron states of a given symmetry, discrete and continuum. In this approach the interactions between the 4f N and the excited configurations via the crystal field potential, the electron correlation operator and the spin–orbit interaction operator are discussed; it is believed that the presented theory contains the most important physical mechanisms responsible for the f↔f electric dipole transitions. Two alternative formulations of the theory of one-photon electric dipole transitions are presented. Consequently, the transition amplitude is defined within the standard theory based on the length formula and within a new approach which is based on the velocity form of the electric dipole radiation operator.

Role of the antenna in tissue selective probes built of lanthanide-organic chelates.

The role of the antenna in the process of the host sensitized luminescence of the DOTA cage coordinated with the Eu ion is investigated. The analysis of the optimal geometries of DOTA modified by several antennas is based on the results of density functional theory (DFT) calculations. The physical environment of the luminescence center (the lanthanide ion) is illustrated by charge density maps and described by the values of the crystal field parameters directly evaluated. The conclusions derived from this theoretical analysis support earlier observations that antennas attached to the cage play the sole role of harvesting and transferring the energy to the lanthanide ion, whereas the cage perturbs the symmetry of the environment of the lanthanide ion, giving rise to the sensitized luminescence. The implications of the separation of the two parts of the organic chelate, cage and antenna, are discussed within the theoretical models of the energy transfer and of forced f <--> f electric dipole transitions.

Relativistic f ↔ f transitions in crystal fields

A relativistic model of f↔f transitions based on the transformation of all intershell tensor operators to effective relativistic double tensor operators is developed. The transition amplitude is expressed in terms of effective operators acting within the fN shell due to a partial closure performed upon the relativistic intershell operators. The final expression is discussed in the light of its reduction to the non-relativistic case that describes f↔f transitions in the language of the standard Judd-Ofelt theory.

Relativistic f ←→ f transitions in crystal fields: II. Beyond the single-configuration approximation

A relativistic model of ff transitions introduced previously is completed here by the third-order contributions caused by electron correlation. The approach is based on the transformation of all tensor operators to their relativistic effective form; the final effective operators that act within the 4f shell are derived by means of the so-called partial closure. The tensorial structure of the new effective operators, that are in general two-particle objects, is discussed and their reduced form is analysed in the light of the standard single-particle parametrization scheme of ff transition spectra.

Physical constraints on sesquiterpene diversity arising from cyclization of the eudesm-5-yl carbocation.

The biogenic origins of complex cyclic terpenes derive from the interplay of enzymes and the intrinsic reactivity of carbocation species at major branch-points along intramolecular cyclization pathways to ultimately determine the distribution of terpene skeletal types in nature. Solanaceous plants biosynthesize chemical defense compounds, largely derived from the eremophilane and spirovetivane-type sesquiterpenes. These hydrocarbon skeletons share a common biogenic origin, stemming from alternative Wagner-Meerwein rearrangements of the eudesm-5-yl carbocation during the cyclization of farnesyl pyrophosphate (FPP) catalyzed by sesquiterpene synthases. While the spirojatamane skeleton shares the same carbocation intermediate, this class of sesquiterpenes has not been reported in the Solanaceae and is exceedingly rare in nature. To investigate the physical basis for alternative rearrangements of the eudesm-5-yl carbocation, we carried out quantum mechanics (QM) analyses to calculate the allowable conformations, energies, and transition states linking conformers of the eudesm-5-yl carbocation to the eremophilene, spirovetivane, and spirojatamane skeletons. Additionally, we conducted parallel investigations on simplified decalin carbocation systems to examine the contribution of ring substituents to allowable conformations and rearrangement pathways. Our study reveals that ring substituents expand the conformational space accessible to the eudesm-5-yl carbocation while sterically blocking rearrangements in certain contexts. From our analysis, we define a conformational threshold for each possible rearrangement based on dihedral angles describing transition state geometry. Further, our calculations indicate that methylene migration rearrangements leading to spiro compounds are thermodynamically dominant in the eudesm-5-yl and simpler decalin carabocation systems. Interestingly, the theoretical abundance of sesquiterpene skeletal types arising from the intrinsic reactivity of the eudesm-5-yl carbocation stands in sharp contrast to their currently known natural abundance. The implications of these results for the catalytic tragectories catalyzed by sesquiterpene synthases are discussed.

Theoretical description of 0 0 and 0 1 transitions in the Eu3+ ion in hosts with C2v symmetry

The results of numerical analysis performed for the electric dipole transitions observed in Eu3+ in the host with C symmetry are presented. The discussion is focused on the one-photon transitions [querry] However, to complete the investigations, numerical results obtained for [querry] are also reported. The approach is based on the second-order Judd-Ofelt theory modified by the third-order terms caused by electron correlation effects. In the case of the 0 1 transition, for the first time, the calculations are performed within the alternative formulation of second-order theory based on the velocity form of electric dipole radiation operator.