Discovery of a new class of bacterial heme-containing CC cleaving oxygenases.

Previously, some bacteria were shown to harbour enzymes capable of catalysing the oxidative cleavage of the double bond of t-anethole and related compounds. The cofactor dependence of these enzymes remained enigmatic due to a lack of biochemical information. We report on catalytic and structural details of a representative of this group of oxidative enzymes: t-anethole oxygenase from Stenotrophomonas maltophilia (TAO).

Biochemical, kinetic, and structural characterization of a Bacillus tequilensis nitroreductase.

Nitroreductases (NRs) are NAD(P)H-dependent flavoenzymes that reduce nitro aromatic compounds to their corresponding arylamines via the nitroso and hydroxylamine intermediates. Because of their broad substrate scope and versatility, NRs have found application in multiple fields such as biocatalysis, bioremediation, cell-imaging and prodrug activation. However, only a limited number of members of the broad NR superfamily (> 24 000 sequences) have been experimentally characterized.

Evolution of the catalytic mechanism at the dawn of the Baeyer-Villiger monooxygenases.

Enzymes are crucial for the emergence and sustenance of life on earth. How they became catalytically active during their evolution is still an open question. Two opposite explanations are plausible: acquiring a mechanism in a series of discrete steps or all at once in a single evolutionary event.

Fixing Flavins: Hijacking a Flavin Transferase for Equipping Flavoproteins with a Covalent Flavin Cofactor.

Most flavin-dependent enzymes contain a dissociable flavin cofactor. We present a new approach for installing in vivo a covalent bond between a flavin cofactor and its host protein. By using a flavin transferase and carving a flavinylation motif in target proteins, we demonstrate that "dissociable" flavoproteins can be turned into covalent flavoproteins.

Characterization of two bacterial multi-flavinylated proteins harboring multiple covalent flavin cofactors.

In recent years, studies have shown that a large number of bacteria secrete multi-flavinylated proteins. The exact roles and properties, of these extracellular flavoproteins that contain multiple covalently anchored FMN cofactors, are still largely unknown. Herein, we describe the biochemical and structural characterization of two multi-FMN-containing covalent flavoproteins, SaFMN3 from and CbFMN4 from .

Regio- and stereoselective steroid hydroxylation by CYP109A2 from Bacillus megaterium explored by X-ray crystallography and computational modeling.

The P450 monooxygenase CYP109A2 from Bacillus megaterium DSM319 was previously found to convert vitamin D3 (VD3) to 25-hydroxyvitamin D3. Here, we show that this enzyme is also able to convert testosterone in a highly regio- and stereoselective manner to 16β-hydroxytestosterone. To reveal the structural determinants governing the regio- and stereoselective steroid hydroxylation reactions catalyzed by CYP109A2, two crystal structures of CYP109A2 were solved in similar closed conformations, one revealing a bound testosterone in the active site pocket, albeit at a nonproductive site away from the heme-iron.

Identification and characterization of archaeal and bacterial F -dependent thioredoxin reductases.

The thioredoxin pathway is an antioxidant system present in most organisms. Electrons flow from a thioredoxin reductase to thioredoxin at the expense of a specific electron donor. Most known thioredoxin reductases rely on NADPH as a reducing cofactor.

Computationally Supported Inversion of Ketoreductase Stereoselectivity.

Whereas directed evolution and rational design by structural inspection are established tools for enzyme redesign, computational methods are less mature but have the potential to predict small sets of mutants with desired properties without laboratory screening of large libraries. We have explored the use of computational enzyme redesign to change the enantioselectivity of a highly thermostable alcohol dehydrogenase from Thermus thermophilus in the asymmetric reduction of ketones. The enzyme reduces acetophenone to (S)-1-phenylethanol.

Computation-Aided Engineering of Cytochrome P450 for the Production of Pravastatin.

CYP105AS1 is a cytochrome P450 from that catalyzes monooxygenation of compactin to 6-epi-pravastatin. For fermentative production of the cholesterol-lowering drug pravastatin, the stereoselectivity of the enzyme needs to be inverted, which has been partially achieved by error-prone PCR mutagenesis and screening. In the current study, we report further optimization of the stereoselectivity by a computationally aided approach.

Substrate Induced Movement of the Metal Cofactor between Active and Resting State.

Regulation of enzyme activity is vital for living organisms. In metalloenzymes, far-reaching rearrangements of the protein scaffold are generally required to tune the metal cofactor's properties by allosteric regulation. Here structural analysis of hydroxyketoacid aldolase from Sphingomonas wittichii RW1 (SwHKA) revealed a dynamic movement of the metal cofactor between two coordination spheres without protein scaffold rearrangements.

A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency.

Soluble pyridine nucleotide transhydrogenases (STHs) are flavoenzymes involved in the redox homeostasis of the essential cofactors NAD(H) and NADP(H). They catalyze the reversible transfer of reducing equivalents between the two nicotinamide cofactors. The soluble transhydrogenase from Escherichia coli (SthA) has found wide use in both in vivo and in vitro applications to steer reducing equivalents toward NADPH-requiring reactions.

Computational Prediction of ω-Transaminase Specificity by a Combination of Docking and Molecular Dynamics Simulations.

ω-Transaminases (ω-TAs) catalyze the conversion of ketones to chiral amines, often with high enantioselectivity and specificity, which makes them attractive for industrial production of chiral amines. Tailoring ω-TAs to accept non-natural substrates is necessary because of their limited substrate range. We present a computational protocol for predicting the enantioselectivity and catalytic selectivity of an ω-TA from with different substrates and benchmark it against 62 compounds gathered from the literature.

Computational Redesign of an ω-Transaminase from for Asymmetric Synthesis of Enantiopure Bulky Amines.

ω-Transaminases (ω-TA) are attractive biocatalysts for the production of chiral amines from prochiral ketones asymmetric synthesis. However, the substrate scope of ω-TAs is usually limited due to steric hindrance at the active site pockets. We explored a protein engineering strategy using computational design to expand the substrate scope of an ()-selective ω-TA from (TA-R6) toward the production of bulky amines.

Catalytic and structural properties of ATP-dependent caprolactamase from Pseudomonas jessenii.

Caprolactamase is the first enzyme in the caprolactam degradation pathway of Pseudomonas jessenii. It is composed of two subunits (CapA and CapB) and sequence-related to other ATP-dependent enzymes involved in lactam hydrolysis, like 5-oxoprolinases and hydantoinases. Low sequence similarity also exists with ATP-dependent acetone- and acetophenone carboxylases.

From thiol-subtilisin to omniligase: Design and structure of a broadly applicable peptide ligase.

Omniligase-1 is a broadly applicable enzyme for peptide bond formation between an activated acyl donor peptide and a non-protected acyl acceptor peptide. The enzyme is derived from an earlier subtilisin variant called peptiligase by several rounds of protein engineering aimed at increasing synthetic yields and substrate range. To examine the contribution of individual mutations on S/H ratio and substrate scope in peptide synthesis, we selected peptiligase variant M222P/L217H as a starting enzyme and introduced successive mutations.

CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering*.

CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation.

Stabilizing AqdC, a Pseudomonas Quinolone Signal-Cleaving Dioxygenase from Mycobacteria, by FRESCO-Based Protein Engineering.

The mycobacterial PQS dioxygenase AqdC, a cofactor-less protein with an α/β-hydrolase fold, inactivates the virulence-associated quorum-sensing signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) produced by the opportunistic pathogen Pseudomonas aeruginosa and is therefore a potential anti-virulence tool. We have used computational library design to predict stabilizing amino acid replacements in AqdC. While 57 out of 91 tested single substitutions throughout the protein led to stabilization, as judged by increases in of >2 °C, they all impaired catalytic activity.

Robust ω-Transaminases by Computational Stabilization of the Subunit Interface.

Transaminases are attractive catalysts for the production of enantiopure amines. However, the poor stability of these enzymes often limits their application in biocatalysis. Here, we used a framework for enzyme stability engineering by computational library design (FRESCO) to stabilize the homodimeric PLP fold type I ω-transaminase from .

Computational Design of Enantiocomplementary Epoxide Hydrolases for Asymmetric Synthesis of Aliphatic and Aromatic Diols.

The use of enzymes in preparative biocatalysis often requires tailoring enzyme selectivity by protein engineering. Herein we explore the use of computational library design and molecular dynamics simulations to create variants of limonene epoxide hydrolase that produce enantiomeric diols from meso-epoxides. Three substrates of different sizes were targeted: cis-2,3-butene oxide, cyclopentene oxide, and cis-stilbene oxide.

Creating Flavin Reductase Variants with Thermostable and Solvent-Tolerant Properties by Rational-Design Engineering.

We have employed computational approaches-FireProt and FRESCO-to predict thermostable variants of the reductase component (C ) of (4-hydroxyphenyl)acetate 3-hydroxylase. With the additional aid of experimental results, two C variants, A166L and A58P, were identified as thermotolerant enzymes, with thermostability improvements of 2.6-5.

Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor.

Metabolic heterogeneity between individual cells of a population harbors significant challenges for fundamental and applied research. Identifying metabolic heterogeneity and investigating its emergence require tools to zoom into metabolism of individual cells. While methods exist to measure metabolite levels in single cells, we lack capability to measure metabolic flux, i.

Efficient Enzymatic Cyclization of Disulfide-Rich Peptides by Using Peptide Ligases.

Disulfide-rich macrocyclic peptides-cyclotides, for example-represent a promising class of molecules with potential therapeutic use. Despite their potential their efficient synthesis at large scale still represents a major challenge. Here we report new chemoenzymatic strategies using peptide ligase variants-inter alia, omniligase-1-for the efficient and scalable one-pot cyclization and folding of the native cyclotides MCoTI-II, kalata B1 and variants thereof, as well as of the θ-defensin RTD-1.

Assessment of the interaction between the flux-signaling metabolite fructose-1,6-bisphosphate and the bacterial transcription factors CggR and Cra.

Bacteria regulate cell physiology in response to extra- and intracellular cues. Recent work showed that metabolic fluxes are reported by specific metabolites, whose concentrations correlate with flux through the respective metabolic pathway. An example of a flux-signaling metabolite is fructose-1,6-bisphosphate (FBP).

Computational redesign of enzymes for regio- and enantioselective hydroamination.

Introduction of innovative biocatalytic processes offers great promise for applications in green chemistry. However, owing to limited catalytic performance, the enzymes harvested from nature's biodiversity often need to be improved for their desired functions by time-consuming iterative rounds of laboratory evolution. Here we describe the use of structure-based computational enzyme design to convert Bacillus sp.

Creating a more robust 5-hydroxymethylfurfural oxidase by combining computational predictions with a novel effective library design.

Background: HMF oxidase (HMFO) from sp. is a recently characterized flavoprotein oxidase. HMFO is a remarkable enzyme as it is able to oxidize 5-hydroxymethylfurfural (HMF) into 2,5-furandicarboxylic acid (FDCA): a catalytic cascade of three oxidation steps.