Paradigms of convergent evolution in enzymes.

There are many occurrences of enzymes catalysing the same reaction but having significantly different structures. Leveraging the comprehensive information on enzymes stored in the Mechanism and Catalytic Site Atlas (M-CSA), we present a collection of 34 cases for which there is sufficient evidence of functional convergence without an evolutionary link. For each case, we compare enzymes which have identical Enzyme Commission numbers (i.

Enzyme function and evolution through the lens of bioinformatics.

Enzymes have been shaped by evolution over billions of years to catalyse the chemical reactions that support life on earth. Dispersed in the literature, or organised in online databases, knowledge about enzymes can be structured in distinct dimensions, either related to their quality as biological macromolecules, such as their sequence and structure, or related to their chemical functions, such as the catalytic site, kinetics, mechanism, and overall reaction. The evolution of enzymes can only be understood when each of these dimensions is considered.

EzMechanism: an automated tool to propose catalytic mechanisms of enzyme reactions.

Over the years, hundreds of enzyme reaction mechanisms have been studied using experimental and simulation methods. This rich literature on biological catalysis is now ripe for use as the foundation of new knowledge-based approaches to investigate enzyme mechanisms. Here, we present a tool able to automatically infer mechanistic paths for a given three-dimensional active site and enzyme reaction, based on a set of catalytic rules compiled from the Mechanism and Catalytic Site Atlas, a database of enzyme mechanisms.

The 3D Modules of Enzyme Catalysis: Deconstructing Active Sites into Distinct Functional Entities.

Enzyme catalysis is governed by a limited toolkit of residues and organic or inorganic co-factors. Therefore, it is expected that recurring residue arrangements will be found across the enzyme space, which perform a defined catalytic function, are structurally similar and occur in unrelated enzymes. Leveraging the integrated information in the Mechanism and Catalytic Site Atlas (M-CSA) (enzyme structure, sequence, catalytic residue annotations, catalysed reaction, detailed mechanism description), 3D templates were derived to represent compact groups of catalytic residues.

Using mechanism similarity to understand enzyme evolution.

Unlabelled: Enzyme reactions take place in the active site through a series of catalytic steps, which are collectively termed the enzyme mechanism. The catalytic step is thereby the individual unit to consider for the purposes of building new enzyme mechanisms - i.e.

AlphaFold2 protein structure prediction: Implications for drug discovery.

The drug discovery process involves designing compounds to selectively interact with their targets. The majority of therapeutic targets for low molecular weight (small molecule) drugs are proteins. The outstanding accuracy with which recent artificial intelligence methods compile the three-dimensional structure of proteins has made protein targets more accessible to the drug design process.

Conformational Variation in Enzyme Catalysis: A Structural Study on Catalytic Residues.

Conformational variation in catalytic residues can be captured as alternative snapshots in enzyme crystal structures. Addressing the question of whether active site flexibility is an intrinsic and essential property of enzymes for catalysis, we present a comprehensive study on the 3D variation of active sites of 925 enzyme families, using explicit catalytic residue annotations from the Mechanism and Catalytic Site Atlas and structural data from the Protein Data Bank. Through weighted pairwise superposition of the functional atoms of active sites, we captured structural variability at single-residue level and examined the geometrical changes as ligands bind or as mutations occur.

GRaSP: a graph-based residue neighborhood strategy to predict binding sites.

Motivation: The discovery of protein-ligand-binding sites is a major step for elucidating protein function and for investigating new functional roles. Detecting protein-ligand-binding sites experimentally is time-consuming and expensive. Thus, a variety of in silico methods to detect and predict binding sites was proposed as they can be scalable, fast and present low cost.

A global analysis of function and conservation of catalytic residues in enzymes.

The catalytic residues of an enzyme comprise the amino acids located in the active center responsible for accelerating the enzyme-catalyzed reaction. These residues lower the activation energy of reactions by performing several catalytic functions. Decades of enzymology research has established general themes regarding the roles of specific residues in these catalytic reactions, but it has been more difficult to explore these roles in a more systematic way.

Exploring Chemical Biosynthetic Design Space with Transform-MinER.

Transform-MinER (Transforming Molecules in Enzyme Reactions) is a web application facilitating the exploration of chemical biosynthetic space, guiding the user toward promising start points for enzyme design projects or directed evolution experiments. Two types of search are possible: Molecule Search allows a user to submit a source substrate enabling Transform-MinER to search for enzyme reactions acting on similar substrates, whereas Path Search additionally allows a user to submit a target molecule enabling Transform-MinER to search for a path of enzyme reactions acting on similar substrates to link source and target. Transform-MinER searches for potential reaction centers in the source substrate and uses chemoinformatic fingerprints to identify those that are situated in molecular environments similar to native counterparts, prioritizing steps that move closer to the target using reactions most similar to native in its exploration of search space.

Finding enzyme cofactors in Protein Data Bank.

Motivation: Cofactors are essential for many enzyme reactions. The Protein Data Bank (PDB) contains >67 000 entries containing enzyme structures, many with bound cofactor or cofactor-like molecules. This work aims to identify and categorize these small molecules in the PDB and make it easier to find them.

Transform-MinER: transforming molecules in enzyme reactions.

Motivation: One goal of synthetic biology is to make new enzymes to generate new products, but identifying the starting enzymes for further investigation is often elusive and relies on expert knowledge, intensive literature searching and trial and error.

Results: We present Transform Molecules in Enzyme Reactions, an online computational tool that transforms query substrate molecules into products using enzyme reactions. The most similar native enzyme reactions for each transformation are found, highlighting those that may be of most interest for enzyme design and directed evolution approaches.

Ranking Enzyme Structures in the PDB by Bound Ligand Similarity to Biological Substrates.

There are numerous applications that use the structures of protein-ligand complexes from the PDB, such as 3D pharmacophore identification, virtual screening, and fragment-based drug design. The structures underlying these applications are potentially much more informative if they contain biologically relevant bound ligands, with high similarity to the cognate ligands. We present a study of ligand-enzyme complexes that compares the similarity of bound and cognate ligands, enabling the best matches to be identified.

Design and synthesis of potent hydroxyethylamine (HEA) BACE-1 inhibitors carrying prime side 4,5,6,7-tetrahydrobenzazole and 4,5,6,7-tetrahydropyridinoazole templates.

A set of low molecular weight compounds containing a hydroxyethylamine (HEA) core structure with different prime side alkyl substituted 4,5,6,7-tetrahydrobenzazoles and one 4,5,6,7-tetrahydropyridinoazole was synthesized. Striking differences were observed on potencies in the BACE-1 enzymatic and cellular assays depending on the nature of the heteroatoms in the bicyclic ring, from the low active compound 4 to inhibitor 6, displaying BACE-1 IC(50) values of 44 nM (enzyme assay) and 65 nM (cell-based assay).

Design and synthesis of potent macrocyclic renin inhibitors.

Two types of P1-P3-linked macrocyclic renin inhibitors containing the hydroxyethylene isostere (HE) scaffold just outside the macrocyclic ring have been synthesized. An aromatic or aliphatic substituent (P3sp) was introduced in the macrocyclic ring aiming at the S3 subpocket (S3sp) in order to optimize the potency. A 5-6-fold improvement in both the K(i) and the human plasma renin activity (HPRA)IC(50) was observed when moving from the starting linear peptidomimetic compound 1 to the most potent macrocycle 42 (K(i) = 3.

Design and synthesis of potent and selective BACE-1 inhibitors.

Highly potent BACE-1 protease inhibitors have been developed from an inhibitors containing a hydroxyethylene (HE) core displaying aryloxymethyl or benzyloxymethyl P1 side chain and a methoxy P1' side chain. The target molecules were synthesized in good overall yields from chiral carbohydrate starting materials. The inhibitors show high BACE-1 potency and good selectivity against cathepsin D, where the most potent inhibitor furnishes BACE-1 K(i) << 1 nM and displays >1000-fold selectivity over cathepsin D.

Synthesis of potent BACE-1 inhibitors incorporating a hydroxyethylene isostere as central core.

We herein describe the design and synthesis of a series of BACE-1 inhibitors incorporating a P1-substituted hydroxylethylene transition state isostere. The synthetic route starting from commercially available carbohydrates yielded a pivotal lactone intermediate with excellent stereochemical control which subsequently could be diversified at the P1-position. The final inhibitors were optimized using three different amines to provide the residues in the P2'-P3' position and three different acids affording the residues in the P2-P3 position.

Catalysing new reactions during evolution: economy of residues and mechanism.

The diversity of function in some enzyme superfamilies shows that during evolution, enzymes have evolved to catalyse different reactions on the same structure scaffold. In this analysis, we examine in detail how enzymes can modify their chemistry, through a comparison of the catalytic residues and mechanisms in 27 pairs of homologous enzymes of totally different functions. We find that evolution is very economical.

Discovery of novel low molecular weight inhibitors of IMPDH via virtual needle screening.

Novel, low molecular weight inhibitors of IMPDH have been discovered through the application of a validated virtual screening protocol. A series of 21 IMPDH inhibitors were used to validate the docking procedure. Application of this procedure to the selection of compounds for screening from an in-house database resulted in a 50-fold reduction in the size of the screening set (3425 to 74 compounds) and gave a hit-rate of 10% on biological evaluation.

Analysis of catalytic residues in enzyme active sites.

We present an analysis of the residues directly involved in catalysis in 178 enzyme active sites. Specific criteria were derived to define a catalytic residue, and used to create a catalytic residue dataset, which was then analysed in terms of properties including secondary structure, solvent accessibility, flexibility, conservation, quaternary structure and function. The results indicate the dominance of a small set of amino acid residues in catalysis and give a picture of a general active site environment.

Structure-based design of peptidomimetic antagonists of p56(lck) SH2 domain.

Starting from the tetrapeptide Ac-pYEEI-NHMe and using a structure-based approach, we have designed and synthesised a peptidomimetic ligand for p56(lck) SH2 domain containing a conformationally restricted replacement for the two glutamate residues. We have explored replacments for the isoleucine residue in the pY+3 pocket and thus identified 1-(R)-amino-3-(S)-indaneacetic acid as the most potent replacement. We also report the X-ray crystal structures of two of the antagonists.