Establishing catalytic activity on an artificial (βα)8-barrel protein designed from identical half-barrels

AbstractIt has been postulated that the ubiquitous (βα)8-barrel enzyme fold has evolved by duplication and fusion of an ancestral (βα)4-half-barrel. We have previously reconstructed this process in the laboratory by fusing two copies of the C-terminal half-barrel HisF-C of imidazole glycerol phosphate synthase (HisF). The resulting construct HisF-CC was stepwise stabilized to Sym1 and Sym2, which are extremely robust but catalytically inert proteins. Here, we report on the generation of a circular permutant of Sym2 and the establishment of a sugar isomerization reaction on its scaffold. Our results demonstrate that duplication and mutagenesis of (βα)4-half-barrels can readily lead to a stable and catalytically active (βα)8-barrel enzyme

2.0 å structure of indole-3-glycerol phosphate synthase from the hyperthermophile Sulfolobus solfataricus: possible determinants of protein stability

AbstractBackground: Recent efforts to understand the basis of protein stability have focussed attention on comparative studies of proteins from hyperthermophilic and mesophilic organisms. Most work to date has been on either oligomeric enzymes or monomers comprising more than one domain. Such studies are hampered by the need to distinguish between stabilizing interactions acting between subunits or domains from those acting within domains. In order to simplify the search for determinants of protein stability we have chosen to study the monomeric enzyme indole-3-glycerol phosphate synthase from the hyperthermophilic archaeon Sulfolobus solfataricus (sIGPS), which grows optimally at 90°C.Results The 2.0 å crystal structure of sIGPS was determined and compared with the known 2.0 å structure of the IGPS domain of the bifunctional enzyme from the mesophilic bacterium Escherichia coli (eIGPS). sIGPS and eIGPS have only 30% sequence identity, but share high structural similarity. Both are single-domain (β/α)8 barrel proteins, with one (eIGPS) or two (sIGPS) additional helices inserted before the first β strand. The thermostable sIGPS has many more salt bridges than eIGPS. Several salt bridges crosslink adjacent α helices or participate in triple or quadruple salt-bridge clusters. The number of helix capping, dipole stabilizing and hydrophobic interactions is also increased in sIGPS.Conclusion The higher stability of sIGPS compared with eIGPS seems to be the result of several improved interactions. These include a larger number of salt bridges, stabilization of α helices and strengthening of both polypeptide chain termini and solvent-exposed loops

Wide-field compensation of monochromatic eye aberrations: expected performance and design trade-offs

Contiene: fórmulas y 6 ilustraciones.The optical quality of the human eye varies across the visual field. Hence an exact compensation of the eye aberration for a given field point can give rise to a less-than-optimum compensation in neighboring field regions. We have studied some aspects of this problem and present here an approach to design wide-field (,10°) optically thin correcting elements, e.g., phase plates, deformable mirrors, and liquid-crystal displays. Their expected performance is assessed using actual eye aberration data. Particular attention is given to the design of elements providing a minimum averaged rms residual aberration and those providing a nearly uniform rms residual aberration across a given field.Work supported by the Spanish Ministerio de Ciencia y Tecnología, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I 1 D 1 I), DPI2002-04370-C02. - This paper was published in Journal of the Optical Society of America A, and is made available as an electronic reprint with the permission of OSA. The paper can be found at the following URL on the OSA website: http://www.opticsinfobase.org/abstract.cfm?URI=josaa-20-1-1. Systematic or multiple reproduction or distribution to multiple locations via electronic or other means is prohibited and is subject to penalties under law.Peer reviewe

TransCent: Computational enzyme design by transferring active sites and considering constraints relevant for catalysis

BACKGROUND: Computational enzyme design is far from being applicable for the general case. Due to computational complexity and limited knowledge of the structure-function interplay, heuristic methods have to be used. RESULTS: We have developed TransCent, a computational enzyme design method supporting the transfer of active sites from one enzyme to an alternative scaffold. In an optimization process, it balances requirements originating from four constraints. These are 1) protein stability, 2) ligand binding, 3) pKa values of active site residues, and 4) structural features of the active site. Each constraint is handled by an individual software module. Modules processing the first three constraints are based on state-of-the-art concepts, i.e. RosettaDesign, DrugScore, and PROPKA. To account for the fourth constraint, knowledge-based potentials are utilized. The contribution of modules to the performance of TransCent was evaluated by means of a recapitulation test. The redesign of oxidoreductase cytochrome P450 was analyzed in detail. As a first application, we present and discuss models for the transfer of active sites in enzymes sharing the frequently encountered triosephosphate isomerase fold. CONCLUSION: A recapitulation test on native enzymes showed that TransCent proposes active sites that resemble the native enzyme more than those generated by RosettaDesign alone. Additional tests demonstrated that each module contributes to the overall performance in a statistically significant manner

Rosetta:MSF: a modular framework for multi-state computational protein design

Computational protein design (CPD) is a powerful technique to engineer existing proteins or to design novel ones that display desired properties. Rosetta is a software suite including algorithms for computational modeling and analysis of protein structures and offers many elaborate protocols created to solve highly specific tasks of protein engineering. Most of Rosetta's protocols optimize sequences based on a single conformation (i.e. design state). However, challenging CPD objectives like multi-specificity design or the concurrent consideration of positive and negative design goals demand the simultaneous assessment of multiple states. This is why we have developed the multi-state framework MSF that facilitates the implementation of Rosetta's single-state protocols in a multi-state environment and made available two frequently used protocols. Utilizing MSF, we demonstrated for one of these protocols that multi-state design yields a 15% higher performance than single-state design on a ligand-binding benchmark consisting of structural conformations. With this protocol, we designed de novo nine retro-aldolases on a conformational ensemble deduced from a (beta alpha)(8)-barrel protein. All variants displayed measurable catalytic activity, testifying to a high success rate for this concept of multi-state enzyme design

Experimental and computational analysis of the ancestry of an evolutionary young enzyme from histidine biosynthesis

The conservation of fold and chemistry of the enzymes associated with histidine biosynthesis suggests that this pathway evolved prior to the diversification of Bacteria, Archaea, and Eukaryotes. The only exception is the histidinol phosphate phosphatase (HolPase). So far, non-homologous HolPases that possess distinct folds and belong to three different protein superfamilies have been identified in various phylogenetic clades. However, their evolution has remained unknown to date. Here, we analyzed the evolutionary history of the HolPase from γ-Proteobacteria (HisB-N). It has been argued that HisB-N and its closest homologue d-glycero-d-manno-heptose-1,7-bisphosphate 7-phosphatase (GmhB) have emerged from the same promiscuous ancestral phosphatase. GmhB variants catalyze the hydrolysis of the anomeric d-glycero-d-manno-heptose-1,7-bisphosphate (αHBP or βHBP) with a strong preference for one anomer (αGmhB or βGmhB). We found that HisB-N from Escherichia coli shows promiscuous activity for βHBP but not αHBP, while βGmhB from Crassaminicella sp. shows promiscuous activity for HolP. Accordingly, a combined phylogenetic tree of αGmhBs, βGmhBs, and HisB-N sequences revealed that HisB-Ns form a compact subcluster derived from βGmhBs. Ancestral sequence reconstruction and in vitro analysis revealed a promiscuous HolPase activity in the resurrected enzymes prior to functional divergence of the successors. The following increase in catalytic efficiency of the HolP turnover is reflected in the shape and electrostatics of the active site predicted by AlphaFold. An analysis of the phylogenetic tree led to a revised evolutionary model that proposes the horizontal gene transfer of a promiscuous βGmhB from δ- to γ-Proteobacteria where it evolved to the modern HisB-N

Evidence for the Existence of Elaborate Enzyme Complexes in the Paleoarchean Era

International audience: Due to the lack of macromolecular fossils, the enzymatic repertoire of extinct species has remained largely unknown to date. In an attempt to solve this problem, we have characterized a cyclase subunit (HisF) of the imidazole glycerol phosphate synthase (ImGP-S), which was reconstructed from the era of the last universal common ancestor of cellular organisms (LUCA). As observed for contemporary HisF proteins, the crystal structure of LUCA-HisF adopts the (βα)8-barrel architecture, one of the most ancient folds. Moreover, LUCA-HisF (i) resembles extant HisF proteins with regard to internal 2-fold symmetry, active site residues, and a stabilizing salt bridge cluster, (ii) is thermostable and shows a folding mechanism similar to that of contemporary (βα)8-barrel enzymes, (iii) displays high catalytic activity, and (iv) forms a stable and functional complex with the glutaminase subunit (HisH) of an extant ImGP-S. Furthermore, we show that LUCA-HisF binds to a reconstructed LUCA-HisH protein with high affinity. Our findings suggest that the evolution of highly efficient enzymes and enzyme complexes has already been completed in the LUCA era, which means that sophisticated catalytic concepts such as substrate channeling and allosteric communication existed already 3.5 billion years ago

Towards Photochromic Azobenzene‐Based Inhibitors for Tryptophan Synthase

Light regulation of drug molecules has gained growing interest in biochemical and pharmacological research in recent years. In addition, a serious need for novel molecular targets of antibiotics has emerged presently. Herein, the development of a photocontrollable, azobenzene‐based antibiotic precursor towards tryptophan synthase (TS), an essential metabolic multienzyme complex in bacteria, is presented. The compound exhibited moderately strong inhibition of TS in its E configuration and five times lower inhibition strength in its Z configuration. A combination of biochemical, crystallographic, and computational analyses was used to characterize the inhibition mode of this compound. Remarkably, binding of the inhibitor to a hitherto‐unconsidered cavity results in an unproductive conformation of TS leading to noncompetitive inhibition of tryptophan production. In conclusion, we created a promising lead compound for combatting bacterial diseases, which targets an essential metabolic enzyme, and whose inhibition strength can be controlled with light

Epimerisation of chiral hydroxylactones by short-chain dehydrogenases/reductases accounts for sex pheromone evolution in Nasonia

Males of all species of the parasitic wasp genus Nasonia use (4R,5S)-5-hydroxy-4-decanolide (RS) as component of their sex pheromone while only N. vitripennis (Nv), employs additionally (4R,5R)-5-hydroxy-4-decanolide (RR). Three genes coding for the NAD(+)-dependent short-chain dehydrogenases/reductases (SDRs) NV10127, NV10128, and NV10129 are linked to the ability of Nv to produce RR. Here we show by assaying recombinant enzymes that SDRs from both Nv and N. giraulti (Ng), the latter a species with only RS in the pheromone, epimerise RS into RR and vice versa with (4R)-5-oxo-4-decanolide as an intermediate. Nv-derived SDR orthologues generally had higher epimerisation rates, which were also influenced by NAD(+) availability. Semiquantitative protein analyses of the pheromone glands by tandem mass spectrometry revealed that NV10127 as well as NV10128 and/or NV10129 were more abundant in Nv compared to Ng. We conclude that the interplay of differential expression patterns and SDR epimerisation rates on the ancestral pheromone component RS accounts for the evolution of a novel pheromone phenotype in Nv