Transferases in Biocatalysis

Enzymes are nature’s catalyst of choice for the highly selective and efficient coupling of carbohydrates. Enzymatic sugar coupling is a competitive technology for industrial glycosylation reactions, since chemical synthetic routes require extensive use of laborious protection group manipulations and often lack regio- and stereoselectivity.BT/Biocatalysi

Enzyme-Catalyzed Synthesis of Esters in Water

MsAcT catalyzes the esterification of primary alcohols in water. When utilizing acid and alcohol as starting materials low yields dictated by thermodynamics were observed. However, with activated esters such as ethyl acetate and vinyl acetate very high yields of the desired ester can be achieved in combination with the appropriate alcohol. This study investigated both the intrinsic kinetic properties of MsAcT for the hydrolysis and transesterification of esters in water as well as the thermodynamics of the reaction. In comparison to the chemical or enzymatic ester synthesis using either toxic reagent, and harsh organic solvents, the MsAcT-catalyzed synthesis of esters of primary alcohols can be achieved efficiently in water without neutralization steps.BT/BiocatalysisEKL Processin

Thermodynamically and Kinetically Controlled Reactions in Biocatalysis – from Concepts to Perspectives

The enzymatic synthesis of esters and peptides is unfavoured in aqueous solvent systems due to competing hydrolysis. This can be overcome by using energy rich substrate analogues: elimination of a good leaving group temporarily establishes more favourable equilibrium conditions, allowing for (nearly) complete conversion. While kinetically controlled syntheses of esters and peptides in water are common knowledge in biocatalysis textbooks, the prevalence of kinetic control is less well known for other enzyme classes. Here, the general concepts of thermodynamic and kinetic control are illustrated at the example of the well-studied synthesis of β-lactam antibiotics and are shown to similarly also apply to other enzyme classes. Notably, the enzymatic synthesis of diastereomers shows the same characteristic energy profile as that of Diels-Alder reactions. This allows for the selective synthesis of different diastereomers under either thermodynamically or kinetically controlled conditions. Prospects and pitfalls of this notion are discussed at the example of the thermodynamic epimerisation of hydroxysteroids and recent examples of kinetically controlled aldol reactions. Kinetic reaction control can therefore not only be used to increase conversions towards a single product, but also to selectively afford different diastereomers. This review highlights the prevalence of both concepts within the field of biocatalysis.BT/Biocatalysi

CH-π Interactions Promote the Conversion of Hydroxypyruvate in a Class II Pyruvate Aldolase

The class II hydroxy ketoacid aldolase A5VH82 from Sphingomonas wittichii RW1 (SwHKA) accepts hydroxypyruvate as nucleophilic donor substrate, giving access to synthetically challenging 3,4-dihydroxy-α-ketoacids. The crystal structure of holo-SwHKA in complex with hydroxypyruvate revealed CH-π interactions between the C−H bonds at C3 of hydroxypyruvate and a phenylalanine residue at position 210, which in this case occupies the position of a conserved leucine residue. Mutagenesis to tyrosine further increased the electron density of the interacting aromatic system and effected a rate enhancement by twofold. While the leucine variant efficiently catalyses the enolisation of hydroxypyruvate as the first step in the aldol reaction, the enol intermediate then becomes trapped in a disfavoured configuration that considerably hinders subsequent C−C bond formation. In SwHKA, micromolar concentrations of inorganic phosphate increase the catalytic rate constant of enolisation by two orders of magnitude. This rate enhancement was now shown to be functionally conserved across the structurally distinct (α/β)8 barrel and αββα sandwich folds of two pyruvate aldolases. Characterisation of the manganese (II) cofactor by electron paramagnetic resonance excluded ionic interactions between the metal centre and phosphate. Instead, histidine 44 was shown to be primarily responsible for the binding of phosphate in the micromolar range and the observed rate enhancement in SwHKA. (Figure presented.).BT/Biocatalysi

Thermodynamics Determine the Diastereochemical Outcome of Catalytic Reactions

Diastereomers are characterised by an intrinsic energy difference, and thermodynamics dictate their distribution within a dynamic equilibrium. The characteristic mechanistic reversibility and non-ideal stereoselectivity of catalysts therefore simultaneously promote both synthesis and epimerization of products during the formation of diastereomers. This feature can even result in the thermodynamic inversion of a chiral centre against the catalyst's stereoselectivity. Here, we provide a comprehensive experimental and theoretical study of factors that govern thermodynamic epimerization in catalysis, using enzymes as example. Our analysis highlights, that the deduction of a catalyst's stereoselectivity based on the absolute configuration of the isolated product constitutes a potential pitfall. The selective formation of either the thermodynamic-, or the kinetic product is less determined by the catalyst, but rather by the reaction conditions. Next to low temperatures, a high maximal extent of conversion was identified to promote kinetically controlled conditions. For bimolecular reactions, conversions can be conveniently modulated via the use of one substrate in excess. Quantum mechanical calculations accurately predicted the diastereomeric excess under equilibrium conditions, which opens the prospect of a rational choice between thermodynamic and kinetic reaction control at an early stage of process design. Our findings are of critical importance for multi-step syntheses of stereocomplex molecules via catalytic cascade reactions or artificial metabolic pathways, as the final stereochemistry may be determined by the absolute configuration of the product that is overall lowest in energy.BT/Biocatalysi

Comparison of Enzymes Immobilised on Immobeads and Inclusion Bodies: A Case Study of a Trehalose Transferase

In this case study, we compare the performance of an enzyme immobilised using two different methods: i) as carrier-free catalytically active inclusion bodies or ii) as carrier-attached immobilised enzyme. To make this comparison we used a trehalose transferase from Thermoproteus uzoniensis fused to the fluorescent thermostable protein mCherry. The fusion of mCherry to trehalose transferase allowed direct spectrophotometric quantification and visualisation of the enzyme in both native and denatured states. The catalytically active inclusion bodies outperformed the immobilised enzyme in their simplicity of biocatalyst production resulting in high enzyme productivity. Enzyme immobilised on carrier materials showed a higher catalytic activity and a more robust performance under batch process conditions.BT/Biocatalysi

A second shell residue modulates a conserved ATP-binding site with radically different affinities for ATP

Background: Prediction of ligand binding and design of new function in enzymes is a time-consuming and expensive process. Crystallography gives the impression that proteins adopt a fixed shape, yet enzymes are functionally dynamic. Molecular dynamics offers the possibility of probing protein movement while predicting ligand binding. Accordingly, we choose the bacterial F1Fo ATP synthase ε subunit to unravel why ATP affinity by ε subunits from Bacillus subtilis and Bacillus PS3 differs ~500-fold, despite sharing identical sequences at the ATP-binding site. Methods: We first used the Bacillus PS3 ε subunit structure to model the B. subtilis ε subunit structure and used this to explore the utility of molecular dynamics (MD) simulations to predict the influence of residues outside the ATP binding site. To verify the MD predictions, point mutants were made and ATP binding studies were employed. Results: MD simulations predicted that E102 in the B. subtilis ε subunit, outside of the ATP binding site, influences ATP binding affinity. Engineering E102 to alanine or arginine revealed a ~10 or ~54 fold increase in ATP binding, respectively, confirming the MD prediction that E102 drastically influences ATP binding affinity. Conclusions: These findings reveal how MD can predict how changes in the “second shell” residues around substrate binding sites influence affinity in simple protein structures. Our results reveal why seemingly identical ε subunits in different ATP synthases have radically different ATP binding affinities. General significance: This study may lead to greater utility of molecular dynamics as a tool for protein design and exploration of protein design and function.BT/Biocatalysi

Leloir Glycosyltransferases in Applied Biocatalysis: A Multidisciplinary Approach

Enzymes are nature's catalyst of choice for the highly selective and efficient coupling of carbohydrates. Enzymatic sugar coupling is a competitive technology for industrial glycosylation reactions, since chemical synthetic routes require extensive use of laborious protection group manipulations and often lack regio- and stereoselectivity. The application of Leloir glycosyltransferases has received considerable attention in recent years and offers excellent control over the reactivity and selectivity of glycosylation reactions with unprotected carbohydrates, paving the way for previously inaccessible synthetic routes. The development of nucleotide recycling cascades has allowed for the efficient production and reuse of nucleotide sugar donors in robust one-pot multi-enzyme glycosylation cascades. In this way, large glycans and glycoconjugates with complex stereochemistry can be constructed. With recent advances, LeLoir glycosyltransferases are close to being applied industrially in multi-enzyme, programmable cascade glycosylations.BT/Biocatalysi

Engineering of continuous bienzymatic cascade process using monolithic microreactors – In flow synthesis of trehalose

Here, we present a two-step continuous flow enzymatic synthesis process in monolithic microreactors using basic sugars as substrates. In the first step UDP-glucose pyrophosphorylase (TaGalU) catalyses the synthesis of uridine-diphosphate-glucose (UDP-Glc) using uridine triphosphate (UTP) and glucose-1-phosphate (Glc-1-P). This is followed by the trehalose transferase (mCherry-TuTreT) catalysed reaction of UDP-Glc and Glc, to obtain trehalose. First, procedures for immobilisation of both enzymes on functionalised silica supports were studied and it was found that covalent bonding by amino groups using a glutaraldehyde linker gives highly active biocatalysts. Due to a drastic difference in temperature range of activity and stability of the immobilised enzymes a bi-reactor cascade was rationally the best solution. Depending on the applied flow rate and hence reaction (residence) time (1.5–10 min) the space-time-yield values varied, respectively, from 1.9 to 14.4 and 8.3 to 49.6 gproduct·L-1·h−1·mgprotein-1, for UDP-glucose pyrophosphorylase and trehalose transferase catalysed reactions. Prolonged (100 h) continuous flow operation showed that the system is operationally stable, but owing to neutral pH, it is prone to microbiological infections. They can be eliminated applying an antibacterial/antifungal therapy or preventive actions by storing and washing the reactors with a NaN3 solution. The presented process paves the way for the continuous in flow synthesis of natural and non-natural trehalose analogues and disaccharides.Green Open Access added to TU Delft Institutional Repository 'You share, we take care!' - Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.BT/Biocatalysi

Substrate Induced Movement of the Metal Cofactor between Active and Resting State

Regulation of enzyme activity is vital for living organisms. In metalloenzymes, far-reaching rearrangements of the protein scaffold are generally required to tune the metal cofactor's properties by allosteric regulation. Here structural analysis of hydroxyketoacid aldolase from Sphingomonas wittichii RW1 (SwHKA) revealed a dynamic movement of the metal cofactor between two coordination spheres without protein scaffold rearrangements. In its resting state configuration (M2+R), the metal constitutes an integral part of the dimer interface within the overall hexameric assembly, but sterical constraints do not allow for substrate binding. Conversely, a second coordination sphere constitutes the catalytically active state (M2+A) at 2.4 Å distance. Bidentate coordination of a ketoacid substrate to M2+A affords the overall lowest energy complex, which drives the transition from M2+R to M2+A. While not described earlier, this type of regulation may be widespread and largely overlooked due to low occupancy of some of its states in protein crystal structures.BT/Biocatalysi