The metal cofactor: stationary or mobile?

Metal cofactors are essential for catalysis and enable countless conversions in nature. Interestingly, the metal cofactor is not always static but mobile with movements of more than 4 Å. These movements of the metal can have different functions. In the case of the xylose isomerase and medium-chain dehydrogenases, it clearly serves a catalytic purpose. The metal cofactor moves during substrate activation and even during the catalytic turnover. On the other hand, in class II aldolases, the enzymes display resting states and active states depending on the movement of the catalytic metal cofactor. This movement is caused by substrate docking, causing the metal cofactor to take the position essential for catalysis. As these metal movements are found in structurally and mechanistically unrelated enzymes, it has to be expected that this metal movement is more common than currently perceived. KEY POINTS: • Metal ions are essential cofactors that can move during catalysis. • In class II aldolases, the metal cofactors can reside in a resting state and an active state. • In MDR, the movement of the metal cofactor is essential for substrate docking.

Biocatalysis making waves in organic chemistry.

Biocatalysis has an enormous impact on chemical synthesis. The waves in which biocatalysis has developed, and in doing so changed our perception of what organic chemistry is, were reviewed 20 and 10 years ago. Here we review the consequences of these waves of development. Nowadays, hydrolases are widely used on an industrial scale for the benign synthesis of commodity and bulk chemicals and are fully developed. In addition, further enzyme classes are gaining ever increasing interest. Particularly, enzymes catalysing selective C-C-bond formation reactions and enzymes catalysing selective oxidation and reduction reactions are solving long-standing synthetic challenges in organic chemistry. Combined efforts from molecular biology, systems biology, organic chemistry and chemical engineering will establish a whole new toolbox for chemistry. Recent developments are critically reviewed.

Synthetic Activity of Recombinant Whole Cell Biocatalysts Containing 2-Deoxy-D-ribose-5-phosphate Aldolase from Pectobacterium atrosepticum.

In nature 2-deoxy-D-ribose-5-phosphate aldolase (DERA) catalyses the reversible formation of 2-deoxyribose 5-phosphate from D-glyceraldehyde 3-phosphate and acetaldehyde. In addition, this enzyme can use acetaldehyde as the sole substrate, resulting in a tandem aldol reaction, yielding 2,4,6-trideoxy-D-erythro-hexapyranose, which spontaneously cyclizes. This reaction is very useful for the synthesis of the side chain of statin-type drugs used to decrease cholesterol levels in blood. One of the main challenges in the use of DERA in industrial processes, where high substrate loads are needed to achieve the desired productivity, is its inactivation by high acetaldehyde concentration. In this work, the utility of different variants of Pectobacterium atrosepticum DERA (PaDERA) as whole cell biocatalysts to synthesize 2-deoxyribose 5-phosphate and 2,4,6-trideoxy-D-erythro-hexapyranose was analysed. Under optimized conditions, E. coli BL21 (PaDERA C-His AA C49M) whole cells yields 99 % of both products. Furthermore, this enzyme is able to tolerate 500 mM acetaldehyde in a whole-cell experiment which makes it suitable for industrial applications.

Methyltransferases: Functions and Applications.

In this review the current state-of-the-art of S-adenosylmethionine (SAM)-dependent methyltransferases and SAM are evaluated. Their structural classification and diversity is introduced and key mechanistic aspects presented which are then detailed further. Then, catalytic SAM as a target for drugs, and approaches to utilise SAM as a cofactor in synthesis are introduced with different supply and regeneration approaches evaluated. The use of SAM analogues are also described. Finally O-, N-, C- and S-MTs, their synthetic applications and potential for compound diversification is given.

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 Šdistance. 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.

Novel oleate hydratases and potential biotechnological applications.

Oleate hydratase catalyses the addition of water to the CC double bond of oleic acid to produce (R)-10-hydroxystearic acid. The enzyme requires an FAD cofactor that functions to optimise the active site structure. A wide range of unsaturated fatty acids can be hydrated at the C10 and in some cases the C13 position. The substrate scope can be expanded using 'decoy' small carboxylic acids to convert small chain alkenes to secondary alcohols, albeit at low conversion rates. Systematic protein engineering and directed evolution to widen the substrate scope and increase the conversion rate is possible, supported by new high throughput screening assays that have been developed. Multi-enzyme cascades allow the formation of a wide range of products including keto-fatty acids, secondary alcohols, secondary amines and α,ω-dicarboxylic acids. KEY POINTS: • Phylogenetically distinct oleate hydratases may exhibit mechanistic differences. • Protein engineering to improve productivity and substrate scope is possible. • Multi-enzymatic cascades greatly widen the product portfolio.

Exploring the abundance of oleate hydratases in the genus Rhodococcus-discovery of novel enzymes with complementary substrate scope.

Oleate hydratases (Ohys, EC 4.2.1.53) are a class of enzymes capable of selective water addition reactions to a broad range of unsaturated fatty acids leading to the respective chiral alcohols. Much research was dedicated to improving the applications of existing Ohys as well as to the identification of undescribed Ohys with potentially novel properties. This study focuses on the latter by exploring the genus Rhodococcus for its plenitude of oleate hydratases. Three different Rhodococcus clades showed the presence of oleate hydratases whereby each clade was represented by a specific oleate hydratase family (HFam). Phylogenetic and sequence analyses revealed HFam-specific patterns amongst conserved amino acids. Oleate hydratases from two Rhodococcus strains (HFam 2 and 3) were heterologously expressed in Escherichia coli and their substrate scope investigated. Here, both enzymes showed a complementary behaviour towards sterically demanding and multiple unsaturated fatty acids. Furthermore, this study includes the characterisation of the newly discovered Rhodococcus pyridinivorans Ohy. The steady-state kinetics of R. pyridinivorans Ohy was measured using a novel coupled assay based on the alcohol dehydrogenase and NAD+-dependent oxidation of 10-hydroxystearic acid.

Assessing the Thiamine Diphosphate Dependent Pyruvate Dehydrogenase E1 Subunit for Carboligation Reactions with Aliphatic Ketoacids.

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 (TKGST). 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.

Artificial Fusion of mCherry Enhances Trehalose Transferase Solubility and Stability.

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.

Rhodococcus as A Versatile Biocatalyst in Organic Synthesis.

The application of purified enzymes as well as whole-cell biocatalysts in synthetic organic chemistry is becoming more and more popular, and both academia and industry are keen on finding and developing novel enzymes capable of performing otherwise impossible or challenging reactions. The diverse genus Rhodococcus offers a multitude of promising enzymes, which therefore makes it one of the key bacterial hosts in many areas of research. This review focused on the broad utilization potential of the genus Rhodococcus in organic chemistry, thereby particularly highlighting the specific enzyme classes exploited and the reactions they catalyze. Additionally, close attention was paid to the substrate scope that each enzyme class covers. Overall, a comprehensive overview of the applicability of the genus Rhodococcus is provided, which puts this versatile microorganism in the spotlight of further research.

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.

2-Deoxy-D-ribose-5-phosphate aldolase (DERA): applications and modifications.

2-Deoxy-D-ribose-5-phosphate aldolase (DERA) is a class I aldolase that offers access to several building blocks for organic synthesis. It catalyzes the stereoselective C-C bond formation between acetaldehyde and numerous other aldehydes. However, the practical application of DERA as a biocatalyst is limited by its poor tolerance towards industrially relevant concentrations of aldehydes, in particular acetaldehyde. Therefore, the development of proper experimental conditions, including protein engineering and/or immobilization on appropriate supports, is required. The present review is aimed to provide a brief overview of DERA, its history, and progress made in understanding the functioning of the enzyme. Furthermore, the current understanding regarding aldehyde resistance of DERA and the various optimizations carried out to modify this property are discussed.

Rhodococcus strains as source for ene-reductase activity.

Rhodococcus strains are ubiquitous in nature and known to metabolise a wide variety of compounds. At the same time, asymmetric reduction of C=C bonds is important in the production of high-valued chiral building blocks. In order to evaluate if Rhodococci can be used for this task, we have probed several Rhodococcus rhodochrous and R. erythropolis strains for ene-reductase activity. A series of substrates including activated ketones, an aldehyde, an imide and nitro-compound were screened using whole cells of seven Rhodococcus strains. This revealed that whole cells of all Rhodococcus strains showed apparent (S)-selectivity towards ketoisophorone, while most other organisms show (R)-selectivity for this compound. Three putative ene-reductases from R. rhodochrous ATCC 17895 were heterologously expressed in Escherichia coli. One protein was purified and its biocatalytic and biochemical properties were characterised, showing typical (enantioselective) properties for class 3 ene-reductases of the old yellow enzyme family.

Enantioselective synthesis of cyanohydrins catalysed by hydroxynitrile lyases - a review.

The first enantioselective synthesis was the selective addition of cyanide to benzaldehyde catalysed by a hydroxynitrile lyase (HNL). Since then these enzymes have been developed into a reliable tool in organic synthesis. HNLs to prepare either the (R)- or the (S)-enantiomer of the desired cyanohydrin are available and a wide variety of reaction conditions can be applied. As a result of this, numerous applications of these enzymes in organic synthesis have been described. Here the examples of the last decade are summarised, the enzyme catalysed step is discussed and the follow-up chemistry is shown. This proves HNLs to be part of main stream organic synthesis. Additionally the newest approaches via immobilisation and reaction engineering are introduced.

Xylella fastidiosa esterase rather than hydroxynitrile lyase.

In 2009, we reported that the product of the gene SCJ21.16 (XFa0032) from Xylella fastidiosa, a xylem-restricted plant pathogen that causes a range of diseases in several important crops, encodes a protein (XfHNL) with putative hydroxynitrile lyase activity. Sequence analysis and activity tests indicated that XfHNL exhibits an α/ß-hydrolase fold and could be classified as a member of the family of FAD-independent HNLs. Here we provide a more detailed sequence analysis and new experimental data. Using pure heterologously expressed XfHNL we show that this enzyme cannot catalyse the cleavage/synthesis of mandelonitrile and that this protein is in fact a non-enantioselective esterase. Homology modelling and ligand docking simulations were used to study the active site and support these results. This finding could help elucidate the common ancestor of esterases and hydroxynitrile lyases with an α/ß -hydrolase fold.

Enantioselective Michael addition of water.

The enantioselective Michael addition using water as both nucleophile and solvent has to date proved beyond the ability of synthetic chemists. Herein, the direct, enantioselective Michael addition of water in water to prepare important ß-hydroxy carbonyl compounds using whole cells of Rhodococcus strains is described. Good yields and excellent enantioselectivities were achieved with this method. Deuterium labeling studies demonstrate that a Michael hydratase catalyzes the water addition exclusively with anti-stereochemistry.

Immobilisation of hydroxynitrile lyases.

Hydroxynitrile lyases are a versatile group of enzymes that are applied both in the laboratory and on an industrial scale. What makes them particularly interesting is that to date five structurally unrelated categories of hydroxynitrile lyases have been discovered. Given their great importance they have often been immobilised utilising many different methodologies. Therefore the hydroxynitrile lyases are ideally suited to compare different immobilisation methods and their dependence on the structural features of the enzyme in question, since the activity is the same in all cases. This review examines all the different immobilisation methods applied to hydroxynitrile lyases and draws conclusions on the effect of the approach.

Enhancement of the substrate scope of transketolase.

To enhance the activity of transketolase towards nonphosphorylated substrates and enlarge the scope of its substrates, notably to long polyol aldehyde acceptors (D-ribose or D-glucose), a rational design-supported evolution strategy was applied. By using docking experiments, an in silico library, and iterative mutagenesis, libraries of single- and double-point mutants were designed and generated. A double-screening approach was implemented, coupling a preselection activity assay (HPLC method) and a selective assay (GC method) to find the best enzymes. Several mutants (R526N, R526Q, R526Q/S525T, R526K/S525T) showed improved activities towards nonphosphorylated substrates as the coupled products of lithium hydroxypyruvate (HPA) with glycolaldehyde (GO), D-ribose or D-glucose. These mutated enzymes were further characterised. They were shown to be up to four times more active than the wild-type (mutant R526Q/S525T) for nonphosphorylated substrates LiHPA/GO (V(m) /K(m) for LiHPA = 92.4 instead of 28.8×10(-3) min(-1) for the wild-type) and 2.6 times more active for substrates LiHPA/rib.

On the synergistic catalytic properties of bimetallic mesoporous materials containing aluminum and zirconium: the Prins cyclisation of citronellal.

Bimetallic three-dimensional amorphous mesoporous materials, Al-Zr-TUD-1 materials, were synthesised by using a surfactant-free, one-pot procedure employing triethanolamine (TEA) as a complexing reagent. The amount of aluminium and zirconium was varied in order to study the effect of these metals on the Brønsted and Lewis acidity, as well as on the resulting catalytic activity of the material. The materials were characterised by various techniques, including elemental analysis, X-ray diffraction, high-resolution TEM, N(2) physisorption, temperature-programmed desorption (TPD) of NH(3), and (27) Al MAS NMR, XPS and FT-IR spectroscopy using pyridine and CO as probe molecules. Al-Zr-TUD-1 materials are mesoporous with surface areas ranging from 700-900 m(2) g(-1), an average pore size of around 4 nm and a pore volume of around 0.70 cm(3) g(-1). The synthesised Al-Zr-TUD-1 materials were tested as catalyst materials in the Lewis acid catalysed Meerwein-Ponndorf-Verley reduction of 4-tert-butylcyclohexanone, the intermolecular Prins synthesis of nopol and in the intramolecular Prins cyclisation of citronellal. Although Al-Zr-TUD-1 catalysts possess a lower amount of acid sites than their monometallic counterparts, according to TPD of NH(3), these materials outperformed those of the monometallic Al-TUD-1 as well as Zr-TUD-1 in the Prins cyclisation of citronellal. This proves the existence of synergistic properties of Al-Zr-TUD-1. Due to the intramolecular nature of the Prins cyclisation of citronellal, the hydrophilic surface of the catalyst as well as the presence of both Brønsted and Lewis acid sites synergy could be obtained with bimetallic Al-Zr-TUD-1. Besides spectroscopic investigation of the active sites of the catalyst material a thorough testing of the catalyst in different types of reactions is crucial in identifying its specific active sites.