Molecular Dynamics Analysis of a Rationally Designed Aldehyde Dehydrogenase Gives Insights into Improved Activity for the Non-Native Cofactor NAD.

The aldehyde dehydrogenase from Thermoplasma acidophilum was previously implemented as a key enzyme in a synthetic cell-free reaction cascade for the production of alcohols. In order to engineer the enzyme's cofactor specificity from NADP+ to NAD+, we identified selectivity-determining residues with the CSR-SALAD tool and investigated further positions based on the crystal structure. Stepwise combination of the initially discovered six point mutations allowed us to monitor the cross effects of each mutation, resulting in a final variant with reduced KM for the non-native cofactor NAD+ (from 18 to 0.6 mM) and an increased activity for the desired substrate d-glyceraldehyde (from 0.4 to 1.5 U/mg). Saturation mutagenesis of the residues at the entrance of the substrate pocket could eliminate substrate inhibition. Molecular dynamics simulations showed a significant gain of flexibility at the cofactor binding site for the final variant. The concomitant increase in stability against isobutanol and only a minor reduction in its temperature stability render the final variant a promising candidate for future optimization of our synthetic cell-free enzymatic cascade.

Development of an Improved Peroxidase-Based High-Throughput Screening for the Optimization of D-Glycerate Dehydratase Activity.

Successful directed evolution examples span a broad range of improved enzyme properties. Nevertheless, the most challenging step for each single directed evolution approach is an efficient identification of improved variants from a large genetic library. Thus, the development and choice of a proper high-throughput screening is a central key for the optimization of enzymes. The detection of low enzymatic activities is especially complicated when they lead to products that are present in the metabolism of the utilized genetic host. Coupled enzymatic assays based on colorimetric products have enabled the optimization of many of such enzymes, but are susceptible to problems when applied on cell extract samples. The purpose of this study was the development of a high-throughput screening for D-glycerate dehydratase activity in cell lysates. With the aid of an automated liquid handling system, we developed a high-throughput assay that relied on a pre-treatment step of cell extract prior to performing the enzymatic and assay reactions. We could successfully apply our method, which should also be transferable to other cell extract-based peroxidase assays, to identify an improved enzyme for the dehydration of D-glycerate.

Optimization of a reduced enzymatic reaction cascade for the production of L-alanine.

Cell-free enzymatic reaction cascades combine the advantages of well-established in vitro biocatalysis with the power of multi-step in vivo pathways. The absence of a regulatory cell environment enables direct process control including methods for facile bottleneck identification and process optimization. Within this work, we developed a reduced, enzymatic reaction cascade for the direct production of L-alanine from D-glucose and ammonium sulfate. An efficient, activity based enzyme selection is demonstrated for the two branches of the cascade. The resulting redox neutral cascade is composed of a glucose dehydrogenase, two dihydroxyacid dehydratases, a keto-deoxy-aldolase, an aldehyde dehydrogenase and an L-alanine dehydrogenase. This artificial combination of purified biocatalysts eliminates the need for phosphorylation and only requires NAD as cofactor. We provide insight into in detail optimization of the process parameters applying a fluorescamine based L-alanine quantification assay. An optimized enzyme ratio and the necessary enzyme load were identified and together with the optimal concentrations of cofactor (NAD), ammonium and buffer yields of >95% for the main branch and of 8% for the side branch were achieved.

Evidence for the existence of elaborate enzyme complexes in the Paleoarchean era.

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 (ßα)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 (ßα)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.

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

It has been postulated that the ubiquitous (ßα)8-barrel enzyme fold has evolved by duplication and fusion of an ancestral (ßα)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 (ßα)4-half-barrels can readily lead to a stable and catalytically active (ßα)8-barrel enzyme.

Conservation of the folding mechanism between designed primordial (ßα)8-barrel proteins and their modern descendant.

The (ßα)(8)-barrel is among the most ancient, frequent, and versatile enzyme structures. It was proposed that modern (ßα)(8)-barrel proteins have evolved from an ancestral (ßα)(4)-half-barrel by gene duplication and fusion. We explored whether the mechanism of protein folding has remained conserved during this long-lasting evolutionary process. For this purpose, potential primordial (ßα)(8)-barrel proteins were constructed by the duplication of a (ßα)(4) element of a modern (ßα)(8)-barrel protein, imidazole glycerol phosphate synthase (HisF), followed by the optimization of the initial construct. The symmetric variant Sym1 was less stable than HisF and its crystal structure showed disorder in the contact regions between the half-barrels. The next generation variant Sym2 was more stable than HisF, and the contact regions were well resolved. Remarkably, both artificial (ßα)(8)-barrels show the same refolding mechanism as HisF and other modern (ßα)(8)-barrel proteins. Early in folding, they all equilibrate rapidly with an off-pathway species. On the productive folding path, they form closely related intermediates and reach the folded state with almost identical rates. The high energy barrier that synchronizes folding is thus conserved. The strong differences in stability between these proteins develop only after this barrier and lead to major changes in the unfolding rates. We conclude that the refolding mechanism of (ßα)(8)-barrel proteins is robust. It evolved early and, apparently, has remained conserved upon the diversification of sequences and functions that have taken place within this large protein family.