Structure-based directed evolution improves S. cerevisiae growth on xylose by influencing in vivo enzyme performance

Background: Efficient bioethanol production from hemicellulose feedstocks by Saccharomyces cerevisiae requires xylose utilization. Whereas S. cerevisiae does not metabolize xylose, engineered strains that express xylose isomerase can metabolize xylose by converting it to xylulose. For this, the type II xylose isomerase from Piromyces (PirXI) is used but the in vivo activity is rather low and very high levels of the enzyme are needed for xylose metabolism. In this study, we explore the use of protein engineering and in vivo selection to improve the performance of PirXI. Recently solved crystal structures were used to focus mutagenesis efforts. Results: We constructed focused mutant libraries of Piromyces xylose isomerase by substitution of second shell residues around the substrate- and metal-binding sites. Following library transfer to S. cerevisiae and selection for enhanced xylose-supported growth under aerobic and anaerobic conditions, two novel xylose isomerase mutants were obtained, which were purified and subjected to biochemical and structural analysis. Apart from a small difference in response to metal availability, neither the new mutants nor mutants described earlier showed significant changes in catalytic performance under various in vitro assay conditions. Yet, in vivo performance was clearly improved. The enzymes appeared to function suboptimally in vivo due to enzyme loading with calcium, which gives poor xylose conversion kinetics. The results show that better in vivo enzyme performance is poorly reflected in kinetic parameters for xylose isomerization determined in vitro with a single type of added metal. Conclusion: This study shows that in vivo selection can identify xylose isomerase mutants with only minor changes in catalytic properties measured under standard conditions. Metal loading of xylose isomerase expressed in yeast is suboptimal and strongly influences kinetic properties. Metal uptake, distribution and binding to xylose isomerase are highly relevant for rapid xylose conversion and may be an important target for optimizing yeast xylose metabolism

Identification and characterization of archaeal and bacterial F420-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 reducing cofactor. Yet, in 2016 a new type of thioredoxin reductase was discovered in archaea which utilizes instead a reduced deazaflavin cofactor (F 420 H 2 ). For this reason, the respective enzyme was named deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR). To have a broader understanding of the biochemistry of DFTRs, we identified and characterized two other archaeal representatives. A detailed kinetic study, which included pre-steady state kinetic analyses, revealed these two DFTRs are highly specific for F 420 H 2 while displaying marginal activity with NADPH. Nevertheless, they share mechanistic features with the canonical thioredoxin reductases that dependent on NADPH (NTRs). A detailed structural analysis led the identification of two key residues that tune cofactor specificity of DFTRs. This allowed us to propose a DFTR-specific sequence motif that enabled for the first time the identification and experimental characterization of a bacterial DFTR. </p

Kinetic and structural properties of a robust bacterial l‐ amino acid oxidase

L-Amino acid oxidase (LAAO) is a flavin adenine dinucleotide (FAD)-dependent enzyme active on most proteinogenic L-amino acids, catalysing their conversion to α-keto acids by oxidative deamination of the substrate. For this oxidation reaction, molecular oxygen is used as the electron acceptor, generating hydrogen peroxide. LAAO can be used to detect L-amino acids, for the production of hydrogen peroxide as an oxidative agent or antimicrobial agent, and for the production of enantiopure amino acids from racemates. In this work, we characterised a previously reported LAAO from the bacterium Pseudoalteromonas luteoviolacea. The substrate scope and kinetic properties of the enzyme were determined, and the thermostability was evaluated. Additionally, we elucidated the crystal structure of this bacterial LAAO, enabling us to test the role of active site residues concerning their function in catalysis. The obtained insights and ease of expression of this thermostable LAAO provides a solid basis for the development of engineered LAAO variants tuned for biosensing and/or biocatalysis

Biochemical properties of a Pseudomonas aminotransferase involved in caprolactam metabolism

The biodegradation of the nylon-6 precursor caprolactam by a strain of Pseudomonas jessenii proceeds via ATP-dependent hydrolytic ring-opening to 6-aminohexanoate. This non-natural ω-amino acid is converted to 6-oxohexanoic acid by an aminotransferase (PjAT) belonging to the fold type I PLP enzymes. To understand the structural basis of 6-aminohexanoatate conversion, we solved different crystal structures and determined the substrate scope with a range of aliphatic and aromatic amines. Comparison with the homologous aminotransferases from Chromobacterium violaceum (CvAT) and Vibrio fluvialis (VfAT) showed that the PjAT enzyme has the lowest KM values (highest affinity) and highest specificity constant (kcat /KM ) with the caprolactam degradation intermediates 6-aminohexanoate and 6-oxohexanoic acid, in accordance with its proposed in vivo function. Five distinct three-dimensional structures of PjAT were solved by protein crystallography. The structure of the aldimine intermediate formed from 6-aminohexanoate and the PLP cofactor revealed the presence of a narrow hydrophobic substrate-binding tunnel leading to the cofactor and covered by a flexible arginine, which explains the high activity and selectivity of the PjAT with 6-aminohexanoate. The results suggest that the degradation pathway for caprolactam has recruited an aminotransferase that is well adapted to 6-aminohexanoate degradation. This article is protected by copyright. All rights reserved

Gene Fusion and Directed Evolution to Break Structural Symmetry and Boost Catalysis by an Oligomeric C‐C Bond‐Forming Enzyme

Gene duplication and fusion are among the primary natural processes that generate new proteins from simpler ancestors. Here we adopted this strategy to evolve a promiscuous homohexameric 4-oxalocrotonate tautomerase (4-OT) into an efficient biocatalyst for enantioselective Michael reactions. We first designed a tandem-fused 4-OT to allow independent sequence diversification of adjacent subunits by directed evolution. This fused 4-OT was then subjected to eleven rounds of directed evolution to give variant 4-OT(F11), which showed an up to 320-fold enhanced activity for the Michael addition of nitromethane to cinnamaldehydes. Crystallographic analysis revealed that 4-OT(F11) has an unusual asymmetric trimeric architecture in which one of the monomers is flipped 180° relative to the others. This gene duplication and fusion strategy to break structural symmetry is likely to become an indispensable asset of the enzyme engineering toolbox, finding wide use in engineering oligomeric proteins

Crystal Structure of Agaricus bisporus Mushroom Tyrosinase: Identity of the Tetramer Subunits and Interaction with Tropolone

Tyrosinase catalyzes the conversion of phenolic compounds into their quinone derivatives, which are precursors for the formation of melanin, a ubiquitous pigment in living organisms. Because of its importance for browning reactions in the food industry, the tyrosinase from the mushroom Agaricus bisporus has been investigated in depth. In previous studies the tyrosinase enzyme complex was shown to be a H2L2 tetramer, but no clues were obtained of the identities of the subunits, their mode of association, and the 3D structure of the complex. Here we unravel this tetramer at the molecular level. Its 2.3 Å resolution crystal structure is the first structure of the full fungal tyrosinase complex. The complex comprises two H subunits of ~392 residues and two L subunits of ~150 residues. The H subunit originates from the ppo3 gene and has a fold similar to other tyrosinases, but it is ~100 residues larger. The L subunit appeared to be the product of orf239342 and has a lectin-like fold. The H subunit contains a binuclear copper-binding site in the deoxy-state, in which three histidine residues coordinate each copper ion. The side chains of these histidines have their orientation fixed by hydrogen bonds or, in the case of His85, by a thioether bridge with the side chain of Cys83. The specific tyrosinase inhibitor tropolone forms a pre-Michaelis complex with the enzyme. It binds near the binuclear copper site without directly coordinating the copper ions. The function of the ORF239342 subunits is not known. Carbohydrate binding sites identified in other lectins are not conserved in ORF239342, and the subunits are over 25 Å away from the active site, making a role in activity unlikely. The structures explain how calcium ions stabilize the tetrameric state of the enzyme.

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. The caprolactamase was produced in Escherichia coli, isolated by His‐tag affinity chromatography, and subjected to functional and structural studies. Activity toward caprolactam required ATP and was dependent on the presence of bicarbonate in the assay buffer. The hydrolysis product was identified as 6‐aminocaproic acid. Quantum mechanical modeling indicated that the hydrolysis of caprolactam was highly disfavored (ΔG(0)'= 23 kJ/mol), which explained the ATP dependence. A crystal structure showed that the enzyme exists as an (αβ)(2) tetramer and revealed an ATP‐binding site in CapA and a Zn‐coordinating site in CapB. Mutations in the ATP‐binding site of CapA (D11A and D295A) significantly reduced product formation. Mutants with substitutions in the metal binding site of CapB (D41A, H99A, D101A, and H124A) were inactive and less thermostable than the wild‐type enzyme. These residues proved to be essential for activity and on basis of the experimental findings we propose possible mechanisms for ATP‐dependent lactam hydrolysis

Structural Elucidation and Engineering of a Bacterial Carbohydrate Oxidase

Flavin-dependent carbohydrate oxidases are valuable tools in biotechnological applications due to their high selectivity in the oxidation of carbohydrates. In this study, we report the biochemical and structural characterization of a recently discovered carbohydrate oxidase from the bacterium Ralstonia solanacearum, which is a member of the vanillyl alcohol oxidase flavoprotein family. Due to its exceptionally high activity toward N-acetyl-d-galactosamine and N-acetyl-d-glucosamine, the enzyme was named N-acetyl-glucosamine oxidase (NagOx). In contrast to most known (fungal) carbohydrate oxidases, NagOx could be overexpressed in a bacterial host, which facilitated detailed biochemical and enzyme engineering studies. Steady state kinetic analyses revealed that non-acetylated hexoses were also accepted as substrates albeit with lower efficiency. Upon determination of the crystal structure, structural insights into NagOx were obtained. A large cavity containing a bicovalently bound FAD, tethered via histidyl and cysteinyl linkages, was observed. Substrate docking highlighted how a single residue (Leu251) plays a key role in the accommodation of N-acetylated sugars in the active site. Upon replacement of Leu251 (L251R mutant), an enzyme variant was generated with a drastically modified substrate acceptance profile, tuned toward non-N-acetylated monosaccharides and disaccharides. Furthermore, the activity toward bulkier substrates such as the trisaccharide maltotriose was introduced by this mutation. Due to its advantage of being overexpressed in a bacterial host, NagOx can be considered a promising alternative engineerable biocatalyst for selective oxidation of monosaccharides and oligosaccharides.</p