Substrate inhibition by the blockage of product release and its control by tunnel engineering.

Substrate inhibition is the most common deviation from Michaelis-Menten kinetics, occurring in approximately 25% of known enzymes. It is generally attributed to the formation of an unproductive enzyme-substrate complex after the simultaneous binding of two or more substrate molecules to the active site. Here, we show that a single point mutation (L177W) in the haloalkane dehalogenase LinB causes strong substrate inhibition. Surprisingly, a global kinetic analysis suggested that this inhibition is caused by binding of the substrate to the enzyme-product complex. Molecular dynamics simulations clarified the details of this unusual mechanism of substrate inhibition: Markov state models indicated that the substrate prevents the exit of the halide product by direct blockage and/or restricting conformational flexibility. The contributions of three residues forming the possible substrate inhibition site (W140A, F143L and I211L) to the observed inhibition were studied by mutagenesis. An unusual synergy giving rise to high catalytic efficiency and reduced substrate inhibition was observed between residues L177W and I211L, which are located in different access tunnels of the protein. These results show that substrate inhibition can be caused by substrate binding to the enzyme-product complex and can be controlled rationally by targeted amino acid substitutions in enzyme access tunnels.

FireProtASR: A Web Server for Fully Automated Ancestral Sequence Reconstruction.

There is a great interest in increasing proteins' stability to widen their usability in numerous biomedical and biotechnological applications. However, native proteins cannot usually withstand the harsh industrial environment, since they are evolved to function under mild conditions. Ancestral sequence reconstruction is a well-established method for deducing the evolutionary history of genes. Besides its applicability to discover the most probable evolutionary ancestors of the modern proteins, ancestral sequence reconstruction has proven to be a useful approach for the design of highly stable proteins. Recently, several computational tools were developed, which make the ancestral reconstruction algorithms accessible to the community, while leaving the most crucial steps of the preparation of the input data on users' side. FireProtASR aims to overcome this obstacle by constructing a fully automated workflow, allowing even the unexperienced users to obtain ancestral sequences based on a sequence query as the only input. FireProtASR is complemented with an interactive, easy-to-use web interface and is freely available at https://loschmidt.chemi.muni.cz/fireprotasr/.

A Haloalkane Dehalogenase from Saccharomonospora viridis Strain DSM 43017, a Compost Bacterium with Unusual Catalytic Residues, Unique (S)-Enantiopreference, and High Thermostability.

Haloalkane dehalogenases can cleave a carbon-halogen bond in a broad range of halogenated aliphatic compounds. However, a highly conserved catalytic pentad composed of a nucleophile, a catalytic base, a catalytic acid, and two halide-stabilizing residues is required for their catalytic activity. Only a few family members, e.g., DsaA, DmxA, or DmrB, remain catalytically active while employing a single halide-stabilizing residue. Here, we describe a novel haloalkane dehalogenase, DsvA, from a mildly thermophilic bacterium, Saccharomonospora viridis strain DSM 43017, possessing one canonical halide-stabilizing tryptophan (W125). At the position of the second halide-stabilizing residue, DsvA contains the phenylalanine F165, which cannot stabilize the halogen anion released during the enzymatic reaction by a hydrogen bond. Based on the sequence and structural alignments, we identified a putative second halide-stabilizing tryptophan (W162) located on the same α-helix as F165, but on the opposite side of the active site. The potential involvement of this residue in DsvA catalysis was investigated by the construction and biochemical characterization of the three variants, DsvA01 (F165W), DsvA02 (W162F), and DsvA03 (W162F and F165W). Interestingly, DsvA exhibits a preference for the (S)- over the (R)-enantiomers of ß-bromoalkanes, which has not been reported before for any characterized haloalkane dehalogenase. Moreover, DsvA shows remarkable operational stability at elevated temperatures. The present study illustrates that protein sequences possessing an unconventional composition of catalytic residues represent a valuable source of novel biocatalysts.IMPORTANCE The present study describes a novel haloalkane dehalogenase, DsvA, originating from a mildly thermophilic bacterium, Saccharomonospora viridis strain DSM 43017. We report its high thermostability, remarkable operational stability at high temperatures, and an (S)-enantiopreference, which makes this enzyme an attractive biocatalyst for practical applications. Sequence analysis revealed that DsvA possesses an unusual composition of halide-stabilizing tryptophan residues in its active site. We constructed and biochemically characterized two single point mutants and one double point mutant and identified the noncanonical halide-stabilizing residue. Our study underlines the importance of searching for noncanonical catalytic residues in protein sequences.

An Ultrasensitive Fluorescence Assay for the Detection of Halides and Enzymatic Dehalogenation.

Halide assays are important for the study of enzymatic dehalogenation, a topic of great industrial and scientific importance. Here we describe the development of a very sensitive halide assay that can detect less than a picomole of bromide ions, making it very useful for quantifying enzymatic dehalogenation products. Halides are oxidised under mild conditions using the vanadium-dependent chloroperoxidase from Curvularia inaequalis, forming hypohalous acids that are detected using aminophenyl fluorescein. The assay is up to three orders of magnitude more sensitive than currently available alternatives, with detection limits of 20 nM for bromide and 1 µM for chloride and iodide. We demonstrate that the assay can be used to determine specific activities of dehalogenases and validate this by comparison to a well-established GC-MS method. This new assay will facilitate the identification and characterisation of novel dehalogenases and may also be of interest to those studying other halide-producing enzymes.

Baeyer-Villiger monooxygenases from Yarrowia lipolytica catalyze preferentially sulfoxidations.

Nine new putative Baeyer-Villiger monooxygenase encoding genes were identified in the eukaryote Yarrowia lipolytica and eight were subsequently cloned and expressed. These enzymes, Yarrowia monooxygenases A-H (YMOA-H), were used in biocatalysis reactions with ketones, sulfides and sulfoxides as substrates. YMOB converts ketones and sulfides, albeit with low activities. However, YMOA did not convert any of the tested ketone substrates, but showed activity towards sulfides and sulfoxides and also showed very high stereoselectivity. This enzyme produced high amounts of sulfones and even converted dimethylsulfoxide (DMSO). Therefore, the sulfoxidation activity of YMOA was investigated in a mutational study. Variants with increased and reduced sulfone yields were created, indicating relevant amino acid positions for the control of sulfoxidation activity. This work expands the set of eukaryotic BVMOs and explores the Yarrowia monooxygenase A, which might belong to a new class of BVMOs as indicated by its unique activity and a phylogenetic analysis.

Hot spots for the protein engineering of Baeyer-Villiger monooxygenases.

Baeyer-Villiger monooxygenases (BVMOs) are versatile biocatalysts for the conversion of ketones to lactones or esters while also being able to efficiently oxidize sulfides to sulfoxides. However, there are limitations for the application of BVMOs in synthesis. In this review we provide an overview of the protein engineering studies aiming at optimizing different properties of BVMOs. We describe hot spots in the active sites of certain BVMOs that have been successfully targeted for changing the substrate scope, as well as the possibility to influence this property by allosteric effects. The identified hot spots in the active sites for controlling enantio- and regioselectivity are shown to be transferable to other BVMOs and we describe concepts to influence heteroatom oxidation, improve protein stability and change the cofactor dependency of BVMOs. Summarizing all these different studies enabled the identification of BVMO- or property-dependent as well as universal hot spots.

Switch in Cofactor Specificity of a Baeyer-Villiger Monooxygenase.

Baeyer-Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to esters or lactones by using molecular oxygen and a cofactor. Type I BVMOs display a strong preference for NADPH. However, for industrial purposes NADH is the preferred cofactor, as it is ten times cheaper and more stable. Thus, we created a variant of the cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMOAcineto ); this used NADH 4200-fold better than NADPH. By combining structure analysis, sequence alignment, and literature data, 21 residues in proximity of the cofactor were identified and targeted for mutagenesis. Two combinatorial variants bearing three or four mutations showed higher conversions of cyclohexanone with NADH (79 %) compared to NADPH (58 %) as well as specificity. The structural reasons for this switch in cofactor specificity of a type I BVMO are especially a hydrogen-bond network coordinating the two hydroxy groups of NADH through direct interactions and bridging water molecules.

Metabolism of alkenes and ketones by Candida maltosa and related yeasts.

Knowledge is scarce about the degradation of ketones in yeasts. For bacteria a subterminal degradation of alkanes to ketones and their further metabolization has been described which always involved Baeyer-Villiger monooxygenases (BVMOs). In addition, the question has to be clarified whether alkenes are converted to ketones, in particular for the oil degrading yeast Candida maltosa little is known. In this study we show the degradation of the aliphatic ketone dodecane-2-one by Candida maltosa and the related yeasts Candida tropicalis, Candida catenulata and Candida albicans as well as Trichosporon asahii and Yarrowia lipolytica. One pathway is initiated by the formation of decyl acetate, resulting from a Baeyer-Villiger-oxidation of this ketone. Beyond this, an initial reduction to dodecane-2-ol by a keto reductase was clearly shown. In addition, two different ways to metabolize dodec-1-ene were proposed. One involved the formation of dodecane-2-one and the other one a conversion leading to carboxylic and dicarboxylic acids. Furthermore the induction of ketone degrading enzymes by dodecane-2-one and dodec-1-ene was shown. Interestingly, with dodecane no subterminal degradation products were detected and it did not induce any enzymes to convert dodecane-2-one.