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 Streptomyces azureus and CbFMN4 from Clostridiaceae bacterium. Based on their primary structure, these proteins were predicted to contain three and four covalently tethered FMN cofactors, respectively. The genes encoding SaFMN3 and CbFMN4 were heterologously coexpressed with a flavin transferase (ApbE) in Escherichia coli, and could be purified by affinity chromatography in good yields. Both proteins were found to be soluble and to contain covalently bound FMN molecules. The SaFMN3 protein was studied in more detail and found to display a single redox potential (-184 mV) while harboring three covalently attached flavins. This is in line with the high sequence similarity when the domains of each flavoprotein are compared. The fully reduced form of SaFMN3 is able to use dioxygen as electron acceptor. Single domains from both proteins were expressed, purified and crystallized. The crystal structures were elucidated, which confirmed that the flavin cofactor is covalently attached to a threonine. Comparison of both crystal structures revealed a high similarity, even in the flavin binding pocket. Based on the crystal structure, mutants of the SaFMN3-D2 domain were designed to improve its fluorescence quantum yield by changing the microenvironment of the isoalloxazine moiety of the flavin cofactor. Residues that quench the flavin fluorescence were successfully identified. Our study reveals biochemical details of multi-FMN-containing proteins, contributing to a better understanding of their role in bacteria and providing leads to future utilization of these flavoprotein in biotechnology.</p

Structure of a robust bacterial protein cage and its application as a versatile biocatalytic platform through enzyme encapsulation

Using a newly discovered encapsulin from Mycolicibacterium hassiacum, several biocatalysts were packaged in this robust protein cage. The encapsulin was found to be easy to produce as recombinant protein. Elucidation of its crystal structure revealed that it is a spherical protein cage of 60 protomers (diameter of 23 nm) with narrow pores. By developing an effective coexpression and isolation procedure, the effect of packaging a variety of biocatalysts could be evaluated. It was shown that encapsulation results in a significantly higher stability of the biocatalysts. Most of the targeted cofactor-containing biocatalysts remained active in the encapsulin. Due to the restricted diameters of the encapsulin pores (5–9 Å), the protein cage protects the encapsulated enzymes from bulky compounds. The work shows that encapsulins may be valuable tools to tune the properties of biocatalysts such as stability and substrate specificity

Critical role of metals in biochemical properties of xylose isomerase

Improving the activity of xylose isomerase (XI) is highly desired for achieving efficient fermentation of xylose in lignocellulosic biomass using XI-expressing S. cerevisiae. XI is a metalloenzyme which requires two bivalent metals for its catalytic activity. The enzyme from Piromyces sp. E2 (PirXI)[1],[2] is activated with various metal ions including Mg2+, Mn2+, Ca2+, Co2+, Zn2+ and Fe2+. The biochemical properties of PirXI are dependent on the types of its metal cofactors. Moreover, the enzyme shows different affinities towards these metals. Characterization of these properties is critical for understanding the enzyme behavior in vivo and to further adapt the enzyme to the cytosolic metal environment. Recently, we have shown that altered intracellular metal composition can improve anaerobic growth of a xylose-fermenting strain by enhancing the activity of PirXI[3]. Furthermore, our current study on PirXI and other studies on different XIs have shown that it is also possible to change the metal preferences of the enzyme[4]. A PirXI variant with a single amino acid substitution in the proximity of the metal binding residues showed significant changes in metal preference compared to the wild-type PirXI. Further exploration on metal specificity of PirXI is necessary to optimize the in vivo enzyme activity. References: 1.Kuyper M, Harhangi HR, Stave AK, Winkler AA, Jetten MS, de Laat WT, den Ridder JJ, Op den Camp HJ, van Dijken JP, Pronk JT, FEMS Yeast Research 4 (2003) 69-78 2.van Maris AJ, Winkler AA, Kuyper M, de Laat WT, van Dijken JP, Pronk JT, Adv Biochem Engin/Biotechnol (2007) 108: 179–204 3.Verhoeven MD, Lee M, Kamoen L, van den Broek M, Janssen DB, Daran JG, van Maris AJA, Pronk JT, Sci. Rep (2017) 7, 46155 4.van Tilbeurgh H, Jenkins J, Chiadmi M, Janin J, Wodak SJ, Mrabet NT, Lambeir AM, Biochemistry (1992) 31: 5467-547

Structure elucidation and characterization of patulin synthase, insights into the formation of a fungal mycotoxin

Patulin synthase (PatE) from Penicillium expansum is a flavin-dependent enzyme that catalyses the last step in the biosynthesis of the mycotoxin patulin. This secondary metabolite is often present in fruit and fruit-derived products, causing postharvest losses. The patE gene was expressed in Aspergillus niger allowing purification and characterization of PatE. This confirmed that PatE is active not only on the proposed patulin precursor ascladiol but also on several aromatic alcohols including 5-hydroxymethylfurfural. By elucidating its crystal structure, details on its catalytic mechanism were revealed. Several aspects of the active site architecture are reminiscent of that of fungal aryl-alcohol oxidases. Yet, PatE is most efficient with ascladiol as substrate confirming its dedicated role in biosynthesis of patulin.</p

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 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 non-productive site away from the heme-iron. To examine if the closed crystal structures nevertheless correspond to a reactive conformation of CYP109A2, docking and molecular dynamics simulations were performed with testosterone and vitamin D3 present in the active site. These molecular dynamics simulations were analyzed for catalytically productive conformations, the relative occurrences of which were in agreement with the experimentally determined stereoselectivities if the predicted stability of each carbon hydrogen bond was taken into account. Overall, the first-time determination and analysis of the catalytically relevant 3D conformation of CYP109A2 will allow for future small molecule ligand screening in silico, as well as enabling site-directed mutagenesis towards improved enzymatic properties of this enzyme.</p

Engineering Thermostability in Artificial Metalloenzymes to Increase Catalytic Activity

Protein engineering has shown widespread use in improving the industrial application of enzymes and broadening the conditions they are able to operate under by increasing their thermostability and solvent tolerance. Here, we show that protein engineering can be used to increase the thermostability of an artificial metalloenzyme. Thermostable variants of the human steroid carrier protein 2L, modified to bind a metal catalyst, were created by rational design using structural data and a 3DM database. These variants were tested to identify mutations that enhanced the stability of the protein scaffold, and a significant increase in melting temperature was observed with a number of modified metalloenzymes. The ability to withstand higher reaction temperatures resulted in an increased activity in the hydroformylation of 1-octene, with more than fivefold improvement in turnover number, whereas the selectivity for linear aldehyde remained high up to 80%

Structural and mutational characterization of the catalytic A-module of the mannuronan C-5-epimerase AlgE4 from Azotobacter vinelandii

Alginate is a family of linear copolymers of (1→4)-linked β-d-mannuronic acid and its C-5 epimer α-l-guluronic acid. The polymer is first produced as polymannuronic acid and the guluronic acid residues are then introduced at the polymer level by mannuronan C-5-epimerases. The structure of the catalytic A-module of the Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 has been determined by x-ray crystallography at 2.1-Å resolution. AlgE4A folds into a right-handed parallel β-helix structure originally found in pectate lyase C and subsequently in several polysaccharide lyases and hydrolases. The β-helix is composed of four parallel β-sheets, comprising 12 complete turns, and has an amphipathic α-helix near the N terminus. The catalytic site is positioned in a positively charged cleft formed by loops extending from the surface encompassing Asp(152), an amino acid previously shown to be important for the reaction. Site-directed mutagenesis further implicates Tyr(149), His(154), and Asp(178) as being essential for activity. Tyr(149) probably acts as the proton acceptor, whereas His(154) is the proton donor in the epimerization reaction

Versatile Peptide C-Terminal Functionalization via a Computationally Engineered Peptide Amidase

The properties of synthetic peptides, including potency, stability, and bioavailability, are strongly influenced by modification of the peptide chain termini. Unfortunately, generally applicable methods for selective and mild C-terminal peptide functionalization are lacking. In this work, we explored the peptide amidase from <i>Stenotrophomonas maltophilia</i> as a versatile catalyst for diverse carboxy-terminal peptide modification reactions. Because the scope of application of the enzyme is hampered by its mediocre stability, we used computational protein engineering supported by energy calculations and molecular dynamics simulations to discover a number of stabilizing mutations. Twelve mutations were combined to yield a highly thermostable (Δ<i><i>T</i></i>_m = 23 °C) and solvent-compatible enzyme. Protein crystallography and molecular dynamics simulations revealed the biophysical effects of mutations contributing to the enhanced robustness. The resulting enzyme catalyzed the selective C-terminal modification of synthetic peptides with small nucleophiles such as ammonia, methylamine, and hydroxylamine in various organic (co)­solvents. The use of a nonaqueous environment allowed modification of peptide free acids with >85% product yield under thermodynamic control. On the basis of the crystal structure, further mutagenesis gave a biocatalyst that favors introduction of larger functional groups. Thus, the use of computational and rational protein design provided a tool for diverse enzymatic peptide modification

Crystallization of the Soluble Lytic Transglycosylase from Escherichia coli K12

Lytic transglycosylases degrade the murein polymer of the bacterial cell wall to 1,6-anhydromuropeptides. These enzymes are of significant medical interest, not only because they are ideal targets for the development of new classes of antibiotics, but also because the low molecular weight products of their catalytic action can cause diverse biological activities in humans, which can be either beneficial or toxic. A soluble lytic transglycosylase was purified from an overproducing Escherichia coli strain and X-ray quality crystals were obtained at room temperature from hanging drops by vapor diffusion against 20 to 25% (NH4)2SO4, in 100 mM-sodium acetate buffer, pH 5.0. The crystals diffract in the X-ray beam to 2.8 Å resolution. Their space group is P212121 with cell dimensions a=81 Å, b=88 Å and c=135 Å. Assuming one monomer (Mr 70,362) per asymmetric unit, the solvent content of these crystals is 63%.