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.

Transketolase catalyzes asymmetric C−C bond formation of two highly polar compounds. Over the last 30 years, the reaction has unanimously been described in literature as irreversible because of the concomitant release of CO₂ if using lithium hydroxypyruvate (LiHPA) as a substrate. Following the reaction over a longer period of time however, we have now found it to be initially kinetically controlled. Contrary to previous suggestions, for the non‐natural conversion of synthetically more interesting apolar substrates, the complete change of active‐site polarity is therefore not necessary. From docking studies it was revealed that water and hydrogen‐bond networks are essential for substrate binding, thus allowing aliphatic aldehydes to be converted in the charged active site of transketolase.

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. magnified image

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.