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.

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.

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.

DERA (2-Deoxy-D-ribose 5-phosphate aldolase) is the only known aldolase that accepts two aldehyde substrates, which makes it an attractive catalyst for the synthesis of a chiral polyol motif that is present in several pharmaceuticals, such as atorvastatin and pravastatin. However, inactivation of the enzyme in the presence of aldehydes hinders its practical application. Whole cells of Pectobacterium atrosepticum were reported to exhibit good tolerance toward acetaldehyde and to afford 2-deoxyribose 5-phosphate with good yields. The DERA gene (PaDERA) was identified, and both the wild-type and a C49M mutant were heterologously expressed in Escherichia coli. The purification protocol was optimized and an initial biochemical characterization was conducted. Unlike other DERAs, which show a maximal activity between pH 4.0 and 7.5, PaDERA presented an optimum pH in the alkaline range between 8.0 and 9.0. This could warrant its use for specific syntheses in the future. PaDERA also displayed fourfold higher specific activity than DERA from E. coli (EcDERA) and displayed a promising acetaldehyde resistance outside the whole-cell environment. The C49M mutation, which was previously identified to increase acetaldehyde tolerance in EcDERA, also led to significant improvements in the acetaldehyde tolerance of PaDERA.

Retaining LeLoir glycosyltransferases catalyze the formation of glycosidic bonds between nucleotide sugar donors and carbohydrate acceptors. The anomeric selectivity of trehalose transferase from Thermoproteus uzoniensis was investigated for both d- and l-glycopyranose acceptors. The enzyme couples a wide range of carbohydrates, yielding trehalose analogues with conversion and enantioselectivity of >98%. The anomeric selectivity inverts from α,α-(1 → 1)-glycosidic bonds for d-glycopyranose acceptors to α,β-(1 → 1)-glycosidic bonds for l-glycopyranose acceptors, while (S)-selectivity was retained for both types of sugar acceptors. Comparison of protein crystal structures of trehalose transferase in complex with α,α-trehalose and an unnatural α,β-trehalose analogue highlighted the mechanistic rationale for the observed inversion of anomeric selectivity.

The synthetic properties of the Thiamine diphosphate (ThDP)-dependent pyruvate dehydrogenase E1 subunit from Escherichia coli (EcPDH E1) was assessed for carboligation reactions with aliphatic ketoacids. Due to its role in metabolism, EcPDH E1 was previously characterised with respect to its biochemical properties, but it was never applied for synthetic purposes. Here, we show that EcPDH E1 is a promising biocatalyst for the production of chiral α-hydroxyketones. WT EcPDH E1 shows a 180–250-fold higher catalytic efficiency towards 2-oxobutyrate or pyruvate, respectively, in comparison to engineered transketolase variants from Geobacillus stearothermophilus (TK_GST). Its broad active site cleft allows for the efficient conversion of both (R)-and (S)-configured α-hydroxyaldehydes, next to linear and branched aliphatic aldehydes as acceptor substrates under kinetically controlled conditions. The alternate, thermodynamically controlled self-reaction of aliphatic aldehydes was shown to be limited to low levels of conversion, which we propose to be due to their large hydration constants. Additionally, the thermodynamically controlled approach was demonstrated to suffer from a loss of stereoselectivity, which makes it unfeasible for aliphatic substrates.

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.

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.

LeLoir glycosyltransferases are important biocatalysts for the production of glycosidic bonds in natural products, chiral building blocks, and pharmaceuticals. Trehalose transferase (TreT) is of particular interest since it catalyzes the stereo- and enantioselective α,α-(1→1) coupling of a nucleotide sugar donor and monosaccharide acceptor for the synthesis of disaccharide derivatives. Heterologously expressed thermophilic trehalose transferases were found to be intrinsically aggregation prone and are mainly expressed as catalytically active inclusion bodies in Escherichia coli To disfavor protein aggregation, the thermostable protein mCherry was explored as a fluorescent protein tag. The fusion of mCherry to trehalose transferase from Pyrobaculum yellowstonensis (PyTreT) demonstrated increased protein solubility. Chaotropic agents like guanidine or the divalent cations Mn(II), Ca(II), and Mg(II) enhanced the enzyme activity of the fusion protein. The thermodynamic equilibrium constant, Keq, for the reversible synthesis of trehalose from glucose and a nucleotide sugar was determined in both the synthesis and hydrolysis directions utilizing UDP-glucose and ADP-glucose, respectively. UDP-glucose was shown to achieve higher conversions than ADP-glucose, highlighting the importance of the choice of nucleotide sugars for LeLoir glycosyltransferases under thermodynamic control.IMPORTANCE The heterologous expression of proteins in Escherichia coli is of great relevance for their functional and structural characterization and applications. However, the formation of insoluble inclusion bodies is observed in approximately 70% of all cases, and the subsequent effects can range from reduced soluble protein yields to a complete failure of the expression system. Here, we present an efficient methodology for the production and analysis of a thermostable, aggregation-prone trehalose transferase (TreT) from Pyrobaculum yellowstonensis via its fusion with mCherry as a thermostable fluorescent protein tag. This fusion strategy allowed for increased enzyme stability and solubility and could be applied to other (thermostable) proteins, allowing rapid visualization and quantification of the mCherry-fused protein of interest. Finally, we have demonstrated that the enzymatic synthesis of trehalose from glucose and a nucleotide sugar is reversible by approaching the thermodynamic equilibrium in both the synthesis and hydrolysis directions. Our results show that uridine establishes an equilibrium constant which is more in favor of the product trehalose than when adenosine is employed as the nucleotide under identical conditions. The influence of different nucleotides on the reaction can be generalized for all LeLoir glycosyltransferases under thermodynamic control as the position of the equilibrium depends solely on the reaction conditions and is not affected by the nature of the catalyst.

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.).