Structural and Functional Insight into the Mechanism of the Fe-S Cluster-Dependent Dehydratase from Paralcaligenes ureilyticus.

Enzyme-catalyzed reaction cascades play an increasingly important role for the sustainable manufacture of diverse chemicals from renewable feedstocks. For instance, dehydratases from the ilvD/EDD superfamily have been embedded into a cascade to convert glucose via pyruvate to isobutanol, a platform chemical for the production of aviation fuels and other valuable materials. These dehydratases depend on the presence of both a Fe-S cluster and a divalent metal ion for their function. However, they also represent the rate-limiting step in the cascade. Here, catalytic parameters and the crystal structure of the dehydratase from Paralcaligenes ureilyticus (PuDHT, both in presence of Mg2+ and Mn2+ ) were investigated. Rate measurements demonstrate that the presence of stoichiometric concentrations Mn2+ promotes higher activity than Mg2+ , but at high concentrations the former inhibits the activity of PuDHT. Molecular dynamics simulations identify the position of a second binding site for the divalent metal ion. Only binding of Mn2+ (not Mg2+ ) to this site affects the ligand environment of the catalytically essential divalent metal binding site, thus providing insight into an inhibitory mechanism of Mn2+ at higher concentrations. Furthermore, in silico docking identified residues that play a role in determining substrate binding and selectivity. The combined data inform engineering approaches to design an optimal dehydratase for the cascade.

Structure-Guided Modulation of the Catalytic Properties of [2Fe-2S]-Dependent Dehydratases.

The FeS cluster-dependent dihydroxyacid dehydratases (DHADs) and sugar acid-specific dehydratases (DHTs) from the ilvD/EDD superfamily are key enzymes in the bioproduction of a wide variety of chemicals. We analyzed [2Fe-2S]-dependent dehydratases in silico and in vitro, deduced functionally relevant sequence, structure, and activity relationships within the ilvD/EDD superfamily, and we propose a new classification based on their evolutionary relationships and substrate profiles. In silico simulations and analyses identified several key positions for specificity, which were experimentally investigated with site-directed and saturation mutagenesis. We thus increased the promiscuity of DHAD from Fontimonas thermophila (FtDHAD), showing >10-fold improved activity toward D-gluconate, and shifted the substrate preference of DHT from Paralcaligenes ureilyticus (PuDHT) toward shorter sugar acids (recording a six-fold improved activity toward the non-natural substrate D-glycerate). The successful elucidation of the role of important active site residues of the ilvD/EDD superfamily will further guide developments of this important biocatalyst for industrial applications.

Dihydroxy-Acid Dehydratases From Pathogenic Bacteria: Emerging Drug Targets to Combat Antibiotic Resistance.

There is an urgent global need for the development of novel therapeutics to combat the rise of various antibiotic-resistant superbugs. Enzymes of the branched-chain amino acid (BCAA) biosynthesis pathway are an attractive target for novel anti-microbial drug development. Dihydroxy-acid dehydratase (DHAD) is the third enzyme in the BCAA biosynthesis pathway. It relies on an Fe-S cluster for catalytic activity and has recently also gained attention as a catalyst in cell-free enzyme cascades. Two types of Fe-S clusters have been identified in DHADs, i.e. [2Fe-2S] and [4Fe-4S], with the latter being more prone to degradation in the presence of oxygen. Here, we characterise two DHADs from bacterial human pathogens, Staphylococcus aureus and Campylobacter jejuni (SaDHAD and CjDHAD). Purified SaDHAD and CjDHAD are virtually inactive, but activity could be reversibly reconstituted in vitro (up to ∼19,000-fold increase with kcat as high as ∼6.7 s-1 ). Inductively-coupled plasma-optical emission spectroscopy (ICP-OES) measurements are consistent with the presence of [4Fe-4S] clusters in both enzymes. N-isopropyloxalyl hydroxamate (IpOHA) and aspterric acid are both potent inhibitors for both SaDHAD (Ki =7.8 and 51.6 µM, respectively) and CjDHAD (Ki =32.9 and 35.1 µM, respectively). These compounds thus present suitable starting points for the development of novel anti-microbial chemotherapeutics.

Characterization of highly active 2-keto-3-deoxy-L-arabinonate and 2-keto-3-deoxy-D-xylonate dehydratases in terms of the biotransformation of hemicellulose sugars to chemicals.

2-keto-3-L-arabinonate dehydratase (L-KdpD) and 2-keto-3-D-xylonate dehydratase (D-KdpD) are the third enzymes in the Weimberg pathway catalyzing the dehydration of respective 2-keto-3-deoxy sugar acids (KDP) to α-ketoglutaric semialdehyde (KGSA). The Weimberg pathway has been explored recently with respect to the synthesis of chemicals from L-arabinose and D-xylose. However, only limited work has been done toward characterizing these two enzymes. In this work, several new L-KdpDs and D-KdpDs were cloned and heterologously expressed in Escherichia coli. Following kinetic characterizations and kinetic stability studies, the L-KdpD from Cupriavidus necator (CnL-KdpD) and D-KdpD from Pseudomonas putida (PpD-KdpD) appeared to be the most promising variants from each enzyme class. Magnesium had no effect on CnL-KdpD, whereas increased activity and stability were observed for PpD-KdpD in the presence of Mg2+. Furthermore, CnL-KdpD was not inhibited in the presence of L-arabinose and L-arabinonate, whereas PpD-KdpD was inhibited with D-xylonate (I50 of 75 mM), but not with D-xylose. Both enzymes were shown to be highly active in the one-step conversions of L-KDP and D-KDP. CnL-KdpD converted > 95% of 500 mM L-KDP to KGSA in the first 2 h while PpD-KdpD converted > 90% of 500 mM D-KDP after 4 h. Both enzymes in combination were able to convert 83% of a racemic mixture of D,L-KDP (500 mM) after 4 h, with both enzymes being specific toward the respective stereoisomer. Key points • L-KdpDs and D-KdpDs are specific toward L- and D-KDP, respectively. • Mg2+affected activity and stabilities of D-KdpDs, but not of L-KdpDs. • CnL-KdpD and PpD-KdpD converted 0.5 M of each KDP isomer reaching 95 and 90% yield. • Both enzymes in combination converted 0.5 M racemic D,L-KDP reaching 83% yield.

Development of a Highly Selective NAD+ -Dependent Glyceraldehyde Dehydrogenase and its Application in Minimal Cell-Free Enzyme Cascades.

Anthropogenic climate change has been caused by over-exploitation of fossil fuels and CO2 emissions. To counteract this, the chemical industry has shifted its focus to sustainable chemical production and the valorization of renewable resources. However, the biggest challenges in biomanufacturing are technical efficiency and profitability. In our minimal cell-free enzyme cascade generating pyruvate as the central intermediate, the NAD+ -dependent, selective oxidation of D-glyceraldehyde was identified as a key reaction step to improve the overall cascade flux. Successive genome mining identified one candidate enzyme with 24-fold enhanced activity and another whose stability is unaffected in 10 % (v/v) ethanol, the final product of our model cascade. Semi-rational engineering improved the substrate selectivity of the enzyme up to 21-fold, thus minimizing side reactions in the one-pot enzyme cascade. The final biotransformation of D-glucose showed a continuous linear production of ethanol (via pyruvate) to a final titer of 4.9 % (v/v) with a molar product yield of 98.7 %. Due to the central role of pyruvate in diverse biotransformations, the optimized production module has great potential for broad biomanufacturing applications.

Hydrogenase-based oxidative biocatalysis without oxygen.

Biocatalysis-based synthesis can provide a sustainable and clean platform for producing chemicals. Many oxidative biocatalytic routes require the cofactor NAD+ as an electron acceptor. To date, NADH oxidase (NOX) remains the most widely applied system for NAD+ regeneration. However, its dependence on O2 implies various technical challenges in terms of O2 supply, solubility, and mass transfer. Here, we present the suitability of a NAD+ regeneration system in vitro based on H2 evolution. The efficiency of the hydrogenase-based system is demonstrated by integrating it into a multi-enzymatic cascade to produce ketoacids from sugars. The total NAD+ recycled using the hydrogenase system outperforms NOX in all different setups reaching up to 44,000 mol per mol enzyme. This system proves to be scalable and superior to NOX in terms of technical simplicity, flexibility, and total output. Furthermore, the system produces only green H2 as a by-product even in the presence of O2.

To beat the heat - engineering of the most thermostable pyruvate decarboxylase to date.

Pyruvate decarboxylase (PDC) is a key enzyme for the production of ethanol at high temperatures and for cell-free butanol synthesis. Thermostable, organic solvent stable PDC was evolved from bacterial PDCs. The new variant shows >1500-fold-improved half-life at 75 °C and >5000-fold-increased half-life in the presence of 9 vol% butanol at 50 °C.

Evaluation of Sequential Processing for the Extraction of Starch, Lipids, and Proteins From Wheat Bran.

In line with the need to better utilize agricultural resources, and valorize underutilized fractions, we have developed protocols to increase the use of wheat bran, to improve utilization of this resource to additional products. Here, we report sequential processing for extraction of starch, lipids, and proteins from wheat brans with two different particle sizes leaving a rest-material enriched in dietary fiber. Mild water-based extraction of starch resulted in maximum 81.7 ± 0.67% yield. Supercritical fluid extraction of lipids by CO2 resulted in 55.2 ± 2.4% yield. This was lower than the corresponding yield using Soxhlet extraction, which was used as a reference method, but allowed a continued extraction sequence without denaturation of the proteins remaining in the raw-material. Alkaline extraction of non-degraded proteins resulted in a yield corresponding to one third of the total protein in the material, which was improved to reach 62 ± 8% by a combination of wheat bran enzymes activation followed by Osborne fractionation. The remaining proteins were extracted in degraded form, resulting in maximum 91.6 ± 1.6% yield of the total proteins content. The remaining material in both fine and coarse bran had a fiber content that on average corresponded to 73 ± 3%. The current work allows separation of several compounds, which is enabling valorization of the bran raw-material into several products.

Structure-Guided Engineering of α-Keto Acid Decarboxylase for the Production of Higher Alcohols at Elevated Temperature.

Branched-chain keto acid decarboxylases (KDCs) are a class of enzymes that catalyze the decarboxylation of α-keto acids. They are key enzymes for production of higher alcohols in vivo and in vitro. However, the two most active KDCs (KivD and KdcA) have only moderate thermostability (<55 °C), which hinders the production of alcohols at high temperatures. Herein, structure-guided engineering toward improved thermostability of KdcA is outlined. Strategies such as stabilization of the catalytic center, surface engineering, and optimization of dimer interactions were applied. With seven amino acid substitutions, variant 7M.D showed an increase of the temperature at which 50 % of activity remains after one-hour incubation T1h50 by 14.8 °C without compromising its substrate specificity. 7M.D exhibited greater than 400-fold improvement of half-life at 70 °C and greater than 600-fold increase in process stability in the presence of 4 % isobutanol at 50 °C. 7M.D is more promising for the production of higher alcohols in thermophiles (>65 °C) and in cell-free applications.