In search for structural targets for engineering d-amino acid transaminase: modulation of pH optimum and substrate specificity.

The development of biocatalysts requires reorganization of the enzyme's active site to facilitate the productive binding of the target substrate and improve turnover number at desired conditions. Pyridoxal-5'-phosphate (PLP) - dependent transaminases are highly efficient biocatalysts for asymmetric amination of ketones and keto acids. However, transaminases, being stereoselective enzymes, have a narrow substrate specificity due to the ordered structure of the active site and work only in neutral-alkaline media. Here, we investigated the d-amino acid transaminase from Aminobacterium colombiense, with the active site organized differently from that of the canonical d-amino acid transaminase from Bacillus sp. YM-1. Using a combination of site-directed mutagenesis, kinetic analysis, molecular modeling, and structural analysis we determined the active site residues responsible for substrate binding, substrate differentiation, thermostability of a functional dimer, and affecting the pH optimum. We demonstrated that the high specificity toward d-glutamate/α-ketoglutarate is due to the interactions of a γ-carboxylate group with K237 residue, while binding of other substrates stems from the effectiveness of their accommodation in the active site optimized for d-glutamate/α-ketoglutarate binding. Furthermore, we showed that the K237A substitution shifts the catalytic activity optimum to acidic pH. Our findings are useful for achieving target substrate specificity and demonstrate the potential for developing and optimizing transaminases for various applications.

Improving Enzyme Fitness with Machine Learning.

The combinatorial composition of proteins has triggered the application of machine learning in enzyme engineering. By predicting how protein sequence encodes function, researchers aim to leverage machine learning models to select a reduced number of optimized sequences for laboratory measurement with the aim to lower costs and shorten timelines of enzyme engineering campaigns. In this review, we will highlight successful algorithm-aided protein engineering examples, including work carried out within the NCCR Catalysis. In this context, we will discuss the underlying computational methods developed to improve enzyme properties such as enantioselectivity, regioselectivity, activity, and stability. Considering the rapid maturing of computational techniques, we expect that their continued application in enzyme engineering campaigns will be key to deliver additional powerful biocatalysts for sustainable chemical synthesis.

Structural insights into oxidation of medium-chain fatty acids and flavanone by myxobacterial cytochrome P450 CYP267B1.

Oxidative biocatalytic reactions performed by cytochrome P450 enzymes (P450s) are of high interest for the chemical and pharmaceutical industries. CYP267B1 is a P450 enzyme from myxobacterium Sorangium cellulosum So ce56 displaying a broad substrate scope. In this work, a search for new substrates was performed, combined with product characterization and a structural analysis of substrate-bound complexes using X-ray crystallography and computational docking. The results demonstrate the ability of CYP267B1 to perform in-chain hydroxylations of medium-chain saturated fatty acids (decanoic acid, dodecanoic acid and tetradecanoic acid) and a regioselective hydroxylation of flavanone. The fatty acids are mono-hydroxylated at different in-chain positions, with decanoic acid displaying the highest regioselectivity towards ω-3 hydroxylation. Flavanone is preferably oxidized to 3-hydroxyflavanone. High-resolution crystal structures of CYP267B1 revealed a very spacious active site pocket, similarly to other P450s capable of converting macrocyclic compounds. The pocket becomes more constricted near to the heme and is closed off from solvent by residues of the F and G helices and the B-C loop. The crystal structure of the tetradecanoic acid-bound complex displays the fatty acid bound near to the heme, but in a nonproductive conformation. Molecular docking allowed modeling of the productive binding modes for the four investigated fatty acids and flavanone, as well as of two substrates identified in a previous study (diclofenac and ibuprofen), explaining the observed product profiles. The obtained structures of CYP267B1 thus serve as a valuable prediction tool for substrate hydroxylations by this highly versatile enzyme and will encourage future selectivity changes by rational protein engineering.

Solvent stable microbial lipases: current understanding and biotechnological applications.

OBJECTIVE: This review examines on our current understanding of microbial lipase solvent tolerance, with a specific focus on the molecular strategies employed to improve lipase stability in a non-aqueous environment. RESULTS: It provides an overview of known solvent tolerant lipases and of approaches to improving solvent stability such as; enhancing stabilising interactions, modification of residue flexibility and surface charge alteration. It shows that judicious selection of lipase source supplemented by appropriate enzyme stabilisation, can lead to a wide application spectrum for lipases. CONCLUSION: Organic solvent stable lipases are, and will continue to be, versatile and adaptable biocatalytic workhorses commonly employed for industrial applications in the food, pharmaceutical and green manufacturing industries.

Novel thermostable amine transferases from hot spring metagenomes.

Hot spring metagenomes, prepared from samples collected at temperatures ranging from 55 to 95 °C, were submitted to an in silico screening aimed at the identification of novel amine transaminases (ATAs), valuable biocatalysts for the preparation of optically pure amines. Three novel (S)-selective ATAs, namely Is3-TA, It6-TA, and B3-TA, were discovered in the metagenome of samples collected from hot springs in Iceland and in Italy, cloned from the corresponding metagenomic DNAs and overexpressed in recombinant form in E. coli. Functional characterization of the novel ATAs demonstrated that they all possess a thermophilic character and are capable of performing amine transfer reactions using a broad range of donor and acceptor substrates, thus suggesting a good potential for practical synthetic applications. In particular, the enzyme B3-TA revealed to be exceptionally thermostable, retaining 85% of activity after 5 days of incubation at 80 °C and more than 40% after 2 weeks under the same condition. These results, which were in agreement with the estimation of an apparent melting temperature around 88 °C, make B3-TA, to the best of our knowledge, the most thermostable natural ATA described to date. This biocatalyst showed also a good tolerance toward different water-miscible and water-immiscible organic solvents. A detailed inspection of the homology-based structural model of B3-TA showed that the overall active site architecture of mesophilic (S)-selective ATAs was mainly conserved in this hyperthermophilic homolog. Additionally, a subfamily of B3-TA-like transaminases, mostly uncharacterized and all from thermophilic microorganisms, was identified and analyzed in terms of phylogenetic relationships and sequence conservation.

The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer-Villiger monooxygenase.

The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer-Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a ß-bulge at the C-terminus of ß-strand 3, which is a feature observed in many proteins of this superfamily.

Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity.

The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor ß-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-aminotransferases.

Copper radical oxidases: galactose oxidase, glyoxal oxidase, and beyond!

The copper radical oxidases (CROs) are an evolutionary and functionally diverse group of enzymes established by the historically significant galactose 6-oxidase and glyoxal oxidase from fungi. Inducted in 2013, CROs now constitute Auxiliary Activity Family 5 (AA5) in the Carbohydrate-Active Enzymes (CAZy) classification. CROs catalyse the two-electron oxidation of their substrates using oxygen as the final electron acceptor and are particularly distinguished by a cross-linked tyrosine-cysteine co-factor that is integral to radical stabilization. Recently, there has been a significant increase in the biochemically and structurally characterized CROs, which has revealed an expanded natural diversity of catalytic activities in the family. This review provides a brief historical introduction to CRO biochemistry and structural biology as a foundation for an update on current advances in CRO enzymology, biotechnology, and biology across kingdoms of life.

Understanding intricacies of bioinspired organic-inorganic hybrid nanoflowers: A quest to achieve enhanced biomolecules immobilization for biocatalytic, biosensing and bioremediation applications.

The immobilization of biomolecules has been a subject of interest for scientists for a long time. The organic-inorganic hybrid nanoflowers are a new class of nanostructures that act as a host platform for the immobilization of such biomolecules. It provides better practical applicability to these functional biomolecules while also providing superior activity and reusability when catalysis is involved. These nanostructures have a versatile and straightforward synthesis process and also exhibit enzyme mimicking activity in many cases. However, this facile synthesis involves many intricacies that require in-depth analysis to fully attain its potential as an immobilization technique. A complete account of all the factors involving the synthesis process optimisation is essential to be studied to make it commercially viable. This paper explores all the different aspects of hybrid nanoflowers which sets them apart from the conventional immobilization techniques while also giving an overview of its wide range of applications in industries.

Structural organization of biocatalytic systems: the next dimension of synthetic metabolism.

In natural metabolic networks, more than 2000 different biochemical reactions are operated and spatially and temporally co-ordinated in a reaction volume of <1 µm3. A similar level of control and precision has not been achieved in chemical synthesis, so far. Recently, synthetic biology succeeded in reconstructing complex synthetic in vitro metabolic networks (SIVMNs) from individual proteins in a defined fashion bottom-up. In this review, we will highlight some examples of SIVMNs and discuss how the further advancement of SIVMNs will require the structural organization of these networks and their reactions to (i) minimize deleterious side reactions, (ii) efficiently energize these networks from renewable energies, and (iii) achieve high productivity. The structural organization of synthetic metabolic networks will be a key step to create novel catalytic systems of the future and advance ongoing efforts of creating cell-like systems and artificial cells.

The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer-Villiger monooxygenase. Corrigendum.

A statement is amended in the article by Isupov et al. [(2015). Acta Cryst. D71, 2344-2353].

New Thermophilic α/ß Class Epoxide Hydrolases Found in Metagenomes From Hot Environments.

Two novel epoxide hydrolases (EHs), Sibe-EH and CH65-EH, were identified in the metagenomes of samples collected in hot springs in Russia and China, respectively. The two α/ß hydrolase superfamily fold enzymes were cloned, over-expressed in Escherichia coli, purified and characterized. The new EHs were active toward a broad range of substrates, and in particular, Sibe-EH was excellent in the desymmetrization of cis-2,3-epoxybutane producing the (2R,3R)-diol product with ee exceeding 99%. Interestingly these enzymes also hydrolyse (4R)-limonene-1,2-epoxide with Sibe-EH being specific for the trans isomer. The Sibe-EH is a monomer in solution whereas the CH65-EH is a dimer. Both enzymes showed high melting temperatures with the CH65-EH being the highest at 85°C retaining 80% of its initial activity after 3 h thermal treatment at 70°C making it the most thermal tolerant wild type epoxide hydrolase described. The Sibe-EH and CH65-EH have been crystallized and their structures determined to high resolution, 1.6 and 1.4 Å, respectively. The CH65-EH enzyme forms a dimer via its cap domains with different relative orientation of the monomers compared to previously described EHs. The entrance to the active site cavity is located in a different position in CH65-EH and Sibe-EH in relation to other known bacterial and mammalian EHs.

Discovery and characterization of thermophilic limonene-1,2-epoxide hydrolases from hot spring metagenomic libraries.

The epoxide hydrolases (EHs) represent an attractive option for the synthesis of chiral epoxides and 1,2-diols which are valuable building blocks for the synthesis of several pharmaceutical compounds. A metagenomic approach has been used to identify two new members of the atypical EH limonene-1,2-epoxide hydrolase (LEH) family of enzymes. These two LEHs (Tomsk-LEH and CH55-LEH) show EH activities towards different epoxide substrates, differing in most cases from those previously identified for Rhodococcus erythropolis (Re-LEH) in terms of stereoselectivity. Tomsk-LEH and CH55-LEH, both from thermophilic sources, have higher optimal temperatures and apparent melting temperatures than Re-LEH. The new LEH enzymes have been crystallized and their structures solved to high resolution in the native form and in complex with the inhibitor valpromide for Tomsk-LEH and poly(ethylene glycol) for CH55-LEH. The structural analysis has provided insights into the LEH mechanism, substrate specificity and stereoselectivity of these new LEH enzymes, which has been supported by mutagenesis studies.

The substrate specificity, enantioselectivity and structure of the (R)-selective amine : pyruvate transaminase from Nectria haematococca.

UNLABELLED: During the last decade the use of transaminases for the production of pharmaceutical and fine chemical intermediates has attracted a great deal of attention. Transaminases are versatile biocatalysts for the efficient production of amine intermediates and many have (S)-enantiospecificity. Transaminases with (R)-specificity are needed to expand the applications of these enzymes in biocatalysis. In this work we have identified a fungal putative (R)-specific transaminase from the Eurotiomycetes Nectria haematococca, cloned a synthetic version of this gene, demonstrated (R)-selective deamination of several substrates including (R)-α-methylbenzylamine, as well as production of (R)-amines, and determined its crystal structure. The crystal structures of the holoenzyme and the complex with an inhibitor gabaculine offer the first detailed insight into the structural basis for substrate specificity and enantioselectivity of the industrially important class of (R)-selective amine : pyruvate transaminases. DATABASE: The atomic coordinates and structure factors for the Nectria TAm in holoenzyme and gabaculine-bound forms have been deposited in the PDB as entries 4cmd and 4cmf respectively.