Channel matters: Overcoming diffusion bottlenecks via loop engineering of LinD for enhanced isoprene production.

Low-energy electron-induced reactions in thin films of glucose and N-acetyl-glucosamine

The development of (bio)catalytic methods to chiral tertiary alcohols is in demand, yet the enantioselective synthesis of tertiary alcohols with sterically hindered moieties is problematic. JanthE is a novel thiamine diphosphate‐dependent enzyme that catalyzes aldehyde‐ketone cross‐coupling reactions with a wide range of donor and acceptor substrates. However, product formation is low and precludes large‐scale production. We investigated the conversion of 2‐oxobutanoate and the bulky ketone phenoxy‐2‐propanone to the product 2‐hydroxy‐2‐methyl‐1‐phenoxypentan‐3‐one as a model reaction for the synthetic capabilities of JanthE. As the reaction design did not significantly improve the yield, we proceeded to rational protein engineering. Docking experiments identified V121, Y268, P293, Y297, and K567 in the active site as important residues for catalysis. Remarkably, the single‐point variant JanthE K567S led to a fivefold increase in product formation (90% ee) compared to the wild type enzyme (93% ee). The reaction was performed at preparative scale proving the direct possibility of application. Additionally, when the reaction was extended to longer 2‐oxopentanoate, the double variant Y297P_K567S showed a 5‐fold increase in product formation compared to the wild type. Our results demonstrate the evolvability and versatility of JanthE for the enantioselective synthesis of sterically hindered tertiary alcohols.

Adrenodoxin (Adx), a [2Fe-2S] vertebrate-type ferredoxin, transfers electrons from the NADPH-dependent flavoprotein Adx reductase (AdR) to mitochondrial cytochrome P450 enzymes of the CYP11A and CYP11B families, which catalyze key reactions in steroid hormone biosynthesis. Adx is a known phosphoprotein, but the kinases that phosphorylate Adx have remained mostly obscure. The aim of this study was to identify previously unknown Adx phosphorylating kinases and to acquire a deeper insight into the functional consequences of such a modification. Here, we show for the first time that bovine Adx is a substrate of protein kinase CK2, whereas bovine CYP11A1, CYP11B1, and AdR are not phosphorylated by this kinase. CK2 phosphorylation of mature Adx requires the presence of both the catalytic alpha-subunit and the regulatory beta-subunit of CK2 and takes place exclusively at residue Thr-71, which is located within the redox partner interaction domain of the protein. We created two Adx mutants, Adx-T71E (imitating a phosphorylation) and Adx-T71V (which cannot be phosphorylated at this site), respectively, and investigated how these mutations affected the interaction of Adx with its redox partners. These data were supplemented with detailed spectroscopic and functional assays using the phosphorylated protein. All Adx species behaved like wild type (Adx-WT) with respect to their redox potential, iron-sulfur cluster symmetry, and overall backbone structure. Substrate conversion assays catalyzed by CYP11A1 showed an increase in product formation when Adx-T71E or CK2-phosphorylated Adx were used as electron carrier instead of Adx-WT, whereas the activity toward CYP11B1 was not altered using these Adx species. Additionally, Adx-T71E represents the only full-length Adx mutant which leads to an increase in CYP11A1 product formation. Therefore, characterizing this full-length mutant helps to improve our knowledge on the functional effects of phosphorylations on complex redox systems.

α+Thalassemia Antagonizes the Malaria-protective Effects of Sickle-Cell Trait

Phenazine methosulfate (PMS) has been successfully used to mediate electron transfer from NADH to cytochrome P-450-CAM in the absence of putidaredoxin and putidaredoxin reductase under aerobic conditions. Identification and quantitation of exo-5- hydroxycamphor , the only product, has been accomplished by gas chromatography. In the absence of cytochrome P-450-CAM, or when other heme proteins (hemoglobin, myoglobin, horseradish peroxidase) are substituted for P-450-CAM, no exo-5- hydroxycamphor is detected. Product formation is not inhibited by the addition of catalase, superoxide dismutase, or hydroxyl radical scavengers; however, significant inhibition is observed with carbon monoxide and metyrapone, known inhibitors of the fully reconstituted P-450 system. Addition of 2,3-dimercaptopropanol to the NADH/PMS/P-450 system leads to a 4-fold increase in product formation; when putidaredoxin is added (without dimercaptopropanol), a 20-fold increase in product formation is observed. Constant bubbling with oxygen results in a further increase in the amount of product (150-fold increase overall). Our results show that PMS can substitute for the electron-transfer proteins putidaredoxin and putidaredoxin reductase in the transfer of electrons from NADH to P-450-CAM, resulting in multiple turnovers. Molecular oxygen dependent multiple turnovers of cytochrome P-450 have not been previously observed without the fully reconstituted, three-protein system.

Bacterial microcompartments (BMCs) enhance the breakdown of metabolites such as 1,2-propanediol (1,2-PD) to propionic acid. The encapsulation of proteins within the BMC is mediated by the presence of targeting sequences. In an attempt to redesign the Pdu BMC into a 1,2-PD synthesising factory using glycerol as the starting material we added N-terminal targeting peptides to glycerol dehydrogenase, dihydroxyacetone kinase, methylglyoxal synthase and 1,2-propanediol oxidoreductase to allow their inclusion into an empty BMC. 1,2-PD producing strains containing the fused enzymes exhibit a 245% increase in product formation in comparison to un-tagged enzymes, irrespective of the presence of BMCs. Tagging of enzymes with targeting peptides results in the formation of dense protein aggregates within the cell that are shown by immuno-labelling to contain the vast majority of tagged proteins. It can therefore be concluded that these protein inclusions are metabolically active and facilitate the significant increase in product formation.

Batch and fed-batch operation result in completely different physiological conditions for cultivated microorganisms or cells. To close the gap between screening, which is hitherto exclusively performed in batch mode, and fed-batch production processes, a special microtiter plate was developed that allows screening in fed-batch mode. The fed-batch microtiter plate (FB-MTP) enables 44 parallel fed-batch experiments at small scale. A small channel filled with a hydrogel connects a reservoir well with a culture well. The nutrient compound diffuses from the reservoir well through the hydrogel into the culture well. Hence, the feed rate can easily be adjusted to the needs of the cultured microorganisms by changing the geometry of the hydrogel channel and the driving concentration gradient. Any desired compound including liquid nutrients like glycerol can be fed to the culture. In combination with an optical measuring device (BioLector), online monitoring of these 44 fed-batch cultures is possible. Two Escherichia coli strains and a Hansenula polymorpha strain were successfully cultivated in the new FB-MTP. As a positive impact of the fed-batch mode on the used strains, a fourfold increase in product formation was observed for E. coli. For H. polymorpha, the use of fed-batch mode resulted in a strong increase in product formation, whereas no measurable product formation was observed in batch mode. In conclusion, the newly developed fed-batch microtiter plate is a versatile, easy-to-use, disposable system to perform fed-batch cultivations at small scale. Screening cultures in high-throughput under online monitoring are possible similar to cultivations under production conditions.

The impact of conformation of the active site loop,secondary structure,active site volume,and substrate(unsaturated acyl chain) channel as a function of simulation time caused by the FabI(enoyl-ACP reductase) inhibitor of triclosan were studied by molecular dynamics simulations,define secondary structure of proteins(DSSP),and pocket volume measurer(POVME).Triclosan restricted the changes of the active site and substrate channel of the FabI- NAD+- TCL(NAD+:nicotinamide adenine dinucleotide,TCL:triclosan) ternary complex.The active site loop formed an ordered,closed,and stable conformation,and was commonly associated with a helical structure in front of the active site.This made the active site volume change little,the volume distribution concentrated and the substrate channel size narrowed and almost closed.However,the active site loop was disordered,open,and flexible in the FabI- NAD+binary complex.The changes of active site volume and distribution in the binary system were larger and more disperse than those in the ternary system.The substrate channel size in the binary system widened and became unstable.Triclosan induced residues of the active site and active site loop of FabI and made the ternary system more closed,which blocked the unsaturated acyl chains from getting into the catalytic center of FabI through the substrate channel,interrupted the reduction reaction and the elongation cycle of fatty acid synthesis.These results aid our understanding of potent inhibitory activity of triclosan and related compounds.

The low energy electron induced damage in self-assembled monolayers of dodecanethiolate, octadecanethiolate, and perdeuterated eicosanethiolate on gold and octadecanethiolate on silver has been investigated in situ by X-ray photoelectron spectroscopy and angle resolved near edge X-ray absorption fine structure spectroscopy. All investigated systems exhibit qualitatively similar behavior with respect to low energy electron irradiation. The most noticeable processes are the loss of orientational and conformational order, partial dehydrogenation with CC double bond formation, desorption of the layer fragments, reduction of the thiolate species, and the appearance of new sulfur species. The cross sections for the rates of the individual irradiation-induced processes have been determined. For the films on gold all these processes are found to evolve with similar rates, except for the formation of CC double bonds and desorption of sulfur-containing fragments. The extent of the latter process is noticeably small...

Cytochrome P450 3A4 (CYP3A4) displays non-Michaelis-Menten kinetics for many of the substrates it metabolizes, including testosterone (TST) and α-naphthoflavone (ANF). Heterotropic effects between these two substrates can further complicate the metabolic profile of the enzyme. In this work, monomeric CYP3A4 solubilized in Nanodiscs has been studied for its ability to interact with varying molar ratios of ANF and TST. Comparison of the observed heme spin state, NADPH consumption, and product formation rates with a non-cooperative model calculated from a linear combination of the global analysis of each substrate reveals a detailed landscape of the heterotropic interactions and indicates negligible binding cooperativity between ANF and TST. The observed effect of ANF on the kinetics of TST metabolism is due to the additive action of the second substrate with no specific allosteric effects.

The Molecular Basis of Substrate Channeling

Tat proteins as novel thylakoid membrane anchors organize a biosynthetic pathway in chloroplasts and increase product yield 5-fold

A carbon−carbon bond formation through cation−anion coupling allows the synthesis of poly(p-phenylenevinylene)s (PPV) 1 and 2a−f, which contain donor and acceptor groups in each vinylene unit. The crucial step in the double bond formation is the quantitative elimination of methanethiol at room temperature. In this work we describe the optimization of the molecular weight of polymers of structure types 1 and 2a−f with regard to (i) solubility of the monomers and polymers and (ii) the reactivity and side reactions of the monomers. The simultaneous donor and acceptor substitution on the central double bond in PPV-analogous structures raises the question of the additivity of dipoles in each repeat unit of a conjugated polymer chain from the viewpoint of potential use in electrooptical applications. The sequence of donor and acceptor groups along the π-conjugated chain can be controlled by the choice of suitable monomers and is crucial for the alignment of the dipole components. The aggregation of polymers 1 a...

Influence of process conditions on end product formation from Clostridium thermocellum 27405 in solid substrate cultivation on paper pulp sludge

CYP105D18 supports H2O2 as an oxygen surrogate for catalysis well and shows high H2O2 resistance capacity. We report the hydroxylation of different steroids using H2O2 as a cosubstrate. Testosterone was regiospecifically hydroxylated to 2β-hydroxytestosterone. Based on the experimental data and molecular docking, we predicted that hydroxylation of methyl testosterone and nandrolone would occur at position 2 in the A-ring, while hydroxylation of androstenedione and adrenosterone was predicted to occur in the B-ring. Further, structure-guided rational design of the substrate access channel was performed with the mutagenesis of residues S63, R82, and F184. Among the mutants, S63A showed a marked decrease in product formation, while F184A showed a significant increase in product formation in testosterone, nandrolone, methyl testosterone, androstenedione, and adrenosterone. The catalytic efficiency (kcat/Km) toward testosterone was increased 1.36-fold in the F184A mutant over that in the wild-type enzyme. These findings might facilitate the potential use of CYP105D18 and further engineering to establish the basis of biotechnological applications. IMPORTANCE The structural modification of steroids is a challenging chemical reaction. Modifying the core ring and the side chain improves the biological activity of steroids. In particular, bacterial cytochrome P450s are used as promiscuous enzymes for the activation of nonreactive carbons of steroids. In the present work, we reported the H2O2-mediated hydroxylation of steroids by CYP105D18, which also overcomes the use of expensive cofactors. Further, exploring the substrate access channel and modifying the bulky amino acid F184A increase substrate conversion while modifying the substrate recognizing amino acid S63 markedly decreases product formation. Exploring the substrate access channel and the rational design of CYP105D18 can improve the substrate conversion, which facilitates the engineering of P450s for industrial application.