Identification, characterization and molecular adaptation of class I redox systems for the production of hydroxylated diterpenoids.

BACKGROUND: De novo production of multi-hydroxylated diterpenoids is challenging due to the lack of efficient redox systems. RESULTS: In this study a new reductase/ferredoxin system from Streptomyces afghaniensis (AfR·Afx) was identified, which allowed the Escherichia coli-based production of the trihydroxylated diterpene cyclooctatin, a potent inhibitor of human lysophospholipase. This production system provides a 43-fold increase in cyclooctatin yield (15 mg/L) compared to the native producer. AfR·Afx is superior in activating the cylcooctatin-specific class I P450s CotB3/CotB4 compared to the conventional Pseudomonas putida derived PdR·Pdx model. To enhance the activity of the PdR·Pdx system, the molecular basis for these activity differences, was examined by molecular engineering. CONCLUSION: We demonstrate that redox system engineering can boost and harmonize the catalytic efficiency of class I hydroxylase enzyme cascades. Enhancing CotB3/CotB4 activities also provided for identification of CotB3 substrate promiscuity and sinularcasbane D production, a functionalized diterpenoid originally isolated from the soft coral Sinularia sp.

Identification of amino acid networks governing catalysis in the closed complex of class I terpene synthases.

Class I terpene synthases generate the structural core of bioactive terpenoids. Deciphering structure-function relationships in the reactive closed complex and targeted engineering is hampered by highly dynamic carbocation rearrangements during catalysis. Available crystal structures, however, represent the open, catalytically inactive form or harbor nonproductive substrate analogs. Here, we present a catalytically relevant, closed conformation of taxadiene synthase (TXS), the model class I terpene synthase, which simulates the initial catalytic time point. In silico modeling of subsequent catalytic steps allowed unprecedented insights into the dynamic reaction cascades and promiscuity mechanisms of class I terpene synthases. This generally applicable methodology enables the active-site localization of carbocations and demonstrates the presence of an active-site base motif and its dominating role during catalysis. It additionally allowed in silico-designed targeted protein engineering that unlocked the path to alternate monocyclic and bicyclic synthons representing the basis of a myriad of bioactive terpenoids.

Predicting the functions and specificity of triterpenoid synthases: a mechanism-based multi-intermediate docking approach.

Terpenoid synthases construct the carbon skeletons of tens of thousands of natural products. To predict functions and specificity of triterpenoid synthases, a mechanism-based, multi-intermediate docking approach is proposed. In addition to enzyme function prediction, other potential applications of the current approach, such as enzyme mechanistic studies and enzyme redesign by mutagenesis, are discussed.

Predicting enzyme-substrate specificity with QM/MM methods: a case study of the stereospecificity of (D)-glucarate dehydratase.

The stereospecificity of d-glucarate dehydratase (GlucD) is explored by QM/MM calculations. Both the substrate binding and the chemical steps of GlucD contribute to substrate specificity. Although the identification of transition states remains computationally intensive, we suggest that QM/MM computations on ground states or intermediates can capture aspects of specificity that cannot be obtained using docking or molecular mechanics methods.

In-silico assessment of protein-protein electron transfer. a case study: cytochrome c peroxidase--cytochrome c.

The fast development of software and hardware is notably helping in closing the gap between macroscopic and microscopic data. Using a novel theoretical strategy combining molecular dynamics simulations, conformational clustering, ab-initio quantum mechanics and electronic coupling calculations, we show how computational methodologies are mature enough to provide accurate atomistic details into the mechanism of electron transfer (ET) processes in complex protein systems, known to be a significant challenge. We performed a quantitative study of the ET between Cytochrome c Peroxidase and its redox partner Cytochrome c. Our results confirm the ET mechanism as hole transfer (HT) through residues Ala194, Ala193, Gly192 and Trp191 of CcP. Furthermore, our findings indicate the fine evolution of the enzyme to approach an elevated turnover rate of 5.47 × 10(6) s(-1) for the ET between Cytc and CcP through establishment of a localized bridge state in Trp191.

Prediction of function for the polyprenyl transferase subgroup in the isoprenoid synthase superfamily.

The number of available protein sequences has increased exponentially with the advent of high-throughput genomic sequencing, creating a significant challenge for functional annotation. Here, we describe a large-scale study on assigning function to unknown members of the trans-polyprenyl transferase (E-PTS) subgroup in the isoprenoid synthase superfamily, which provides substrates for the biosynthesis of the more than 55,000 isoprenoid metabolites. Although the mechanism for determining the product chain length for these enzymes is known, there is no simple relationship between function and primary sequence, so that assigning function is challenging. We addressed this challenge through large-scale bioinformatics analysis of >5,000 putative polyprenyl transferases; experimental characterization of the chain-length specificity of 79 diverse members of this group; determination of 27 structures of 19 of these enzymes, including seven cocrystallized with substrate analogs or products; and the development and successful application of a computational approach to predict function that leverages available structural data through homology modeling and docking of possible products into the active site. The crystallographic structures and computational structural models of the enzyme-ligand complexes elucidate the structural basis of specificity. As a result of this study, the percentage of E-PTS sequences similar to functionally annotated ones (BLAST e-value ≤ 1e(-70)) increased from 40.6 to 68.8%, and the percentage of sequences similar to available crystal structures increased from 28.9 to 47.4%. The high accuracy of our blind prediction of newly characterized enzymes indicates the potential to predict function to the complete polyprenyl transferase subgroup of the isoprenoid synthase superfamily computationally.

QM/MM methods: looking inside heme proteins biochemistry.

Mixed quantum mechanics/molecular mechanics methods offer a valuable computational tool for understanding biochemical events. When combined with conformational sampling techniques, they allow for an exhaustive exploration of the enzymatic mechanism. Heme proteins are ubiquitous and essential for every organism. In this review we summarize our efforts towards the understanding of heme biochemistry. We present: 1) results on ligand migration on globins coupled to the ligand binding event, 2) results on the localization of the spin density in compound I of cytochromes and peroxidases, 3) novel methodologies for mapping the electron transfer pathways and 4) novel data on Tryptophan 2,3-dioxygenase. For this enzyme our results strongly indicate that the distal oxygen will end up on the C3 indole carbon, whereas the proximal oxygen will end up in the C2 position. Interestingly, the process involves the formation of an epoxide and a heme ferryl intermediate. The overall energy profile indicates an energy barrier of approximately 18 kcal/mol and an exothermic driving force of almost 80 kcal/mol.

Temperature Effects on Donor-Acceptor Couplings in Peptides. A Combined Quantum Mechanics and Molecular Dynamics Study.

We report a quantum chemistry and molecular dynamics study on the temperature dependence of electronic coupling in two short model oligopeptides. Ten nanoseconds replica exchange molecular dynamics was performed on Trp-(Pro)3-Trp and Trp-(Pro)6-Trp peptides in the gas phase in combination with computation of the energy and electronic coupling for thermal hole transfer between Trp residues. The electron transfer parameters were estimated by using the semiempirical INDO/S method together with the charge fragment difference scheme. Conformational analysis of the derived trajectories revealed that the electronic coupling becomes temperature dependent when incorporating structural dynamics of the system. We demonstrate that Trp-(Pro)3-Trp, having only few degrees of freedom, results in relatively weak couplings at low and high temperature and a strong peak at 144 K, whereas the more flexible system Trp-(Pro)6-Trp shows monotonically decreased coupling. Only a few conformations with strong donor-acceptor couplings are shown to be crucial for the overall ET rates. Our results introduce the question whether the T dependence of ET coupling can also be found in large biological systems.

Solvent Effects on Donor-Acceptor Couplings in Peptides. A Combined QM and MD Study.

We present a combined Quantum Chemical/Molecular Dynamics study on electronic coupling between tryptophan-based donor and acceptor in oligopeptides of variable length. Molecular dynamics was performed on Trp-(Pro)n-Trp (n = 1 to 6) molecules in gas phase and aqueous solvent and the electronic coupling matrix element was computed for thermal hole transfer applying semiempirical INDO/S together with the generalized Mulliken-Hush approach. For comparison, we also computed coupling values of 40 000 snapshots applying ab initio Hartree-Fock, showing good agreement with the INDO/S results. We demonstrate that the coupling values strongly fluctuate throughout the molecular dynamic trajectory and the mechanism of electron transfer is affected by the presence of solvent through restriction of the conformational space. Gas-phase calculations show gated electron transfer dominated by direct through-space coupling due to strong conformational changes bringing donor and acceptor in close vicinity. Solvent calculations establish a nongated mechanism dominated by bridge-mediated coupling. In agreement with experimental data, our results point to a donor-acceptor distance of ∼20 Šas a possible point for transition from superexchange to hopping electron transfer mechanism.

Electron transfer in the P450cam/PDX complex. The QM/MM e-pathway.

Electron transfer processes are simple but crucial reactions in biochemistry, being one of the main steps in almost all enzymatic cycles. Obtaining an atomic description of the transfer pathway is a difficult task, at both the experimental and theoretical levels. Here we combine protein-protein docking algorithms, protein structure prediction methodologies and mixed quantum mechanics/molecular mechanics techniques to map the electron transfer pathway between cytochrome P450 camphor and its redox partner, putidaredoxin. Although the mechanism of interaction and electron transfer for this redox couple has been under investigation for over 30 years, the exact mechanism and electron transfer pathway has not been fully understood, yet. Our results report the first ab initio quantum chemistry description of the electron migration. The obtained electron transfer pathway indicates the key role of Arg112 of P450 and Asp38 of PDX and the existence of slightly different electron transfer pathways for different protein-protein complexes.

Mapping protein electron transfer pathways with QM/MM methods.

Mixed quantum mechanics/molecular mechanics (QM/MM) methods offer a valuable computational tool for understanding the electron transfer pathway in protein-substrate interactions and protein-protein complexes. These hybrid methods are capable of solving the Schrödinger equation on a small subset of the protein, the quantum region, describing its electronic structure under the polarization effects of the remainder of the protein. By selectively turning on and off different residues in the quantum region, we are able to obtain the electron pathway for short- and large-range interactions. Here, we summarize recent studies involving the protein-substrate interaction in cytochrome P450 camphor, ascorbate peroxidase and cytochrome c peroxidase, and propose a novel approach for the long-range protein-protein electron transfer. The results on ascorbate peroxidase and cytochrome c peroxidase reveal the importance of the propionate groups in the electron transfer pathway. The long-range protein-protein electron transfer has been studied on the cytochrome c peroxidase-cytochrome c complex. The results indicate the importance of Phe82 and Cys81 on cytochrome c, and of Asn196, Ala194, Ala176 and His175 on cytochrome c peroxidase.