Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S)-selectivity and together with a highly (R)-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases.

The glucose-methanol-choline (GMC) superfamily is a large and functionally diverse family of oxidoreductases that share a common structural fold. Fungal members of this superfamily that are characterised and relevant for lignocellulose degradation include aryl-alcohol oxidoreductase, alcohol oxidase, cellobiose dehydrogenase, glucose oxidase, glucose dehydrogenase, pyranose dehydrogenase, and pyranose oxidase, which together form family AA3 of the auxiliary activities in the CAZy database of carbohydrate-active enzymes. Overall, little is known about the extant sequence space of these GMC oxidoreductases and their phylogenetic relations. Although some individual forms are well characterised, it is still unclear how they compare in respect of the complete enzyme class and, therefore, also how generalizable are their characteristics.

The use of oxidoreductases (EC1) in non-conventional reaction media has been increasingly explored. In particular, deep eutectic solvents (DESs) have emerged as a novel class of solvents. Herein, an in-depth study of bioreduction with an alcohol dehydrogenase (ADH) in the DES glyceline is presented. The activity and stability of ADH in mixtures of glyceline/water with varying water contents were measured. Furthermore, the thermodynamic water activity and viscosity of mixtures of glyceline/water have been determined. For a better understanding of the observations, molecular dynamics simulations were performed to quantify the molecular flexibility, hydration layer, and intraprotein hydrogen bonds of ADH. The behavior of the enzyme in DESs follows the classic dependence of water activity (aW ) in non-conventional media. At low aW values (<0.2), ADH does not show any activity; at higher aW values, the activity was still lower than that in pure water due to the high viscosities of the DES. These findings could be further explained by increased enzyme flexibility with increasing water content.

The first step in methanol metabolism in methylotrophic yeasts, the oxidation of methanol and higher alcohols with molecular oxygen to formaldehyde and hydrogen peroxide, is catalysed by alcohol oxidase (AOX), a 600-kDa homo-octamer containing eight FAD cofactors. When these yeasts are grown with methanol as the carbon source, AOX forms large crystalline arrays in peroxisomes. We determined the structure of AOX by cryo-electron microscopy at a resolution of 3.4 Å. All residues of the 662-amino acid polypeptide as well as the FAD are well resolved. AOX shows high structural homology to other members of the GMC family of oxidoreductases, which share a conserved FAD binding domain, but have different substrate specificities. The preference of AOX for small alcohols is explained by the presence of conserved bulky aromatic residues near the active site. Compared to the other GMC enzymes, AOX contains a large number of amino acid inserts, the longest being 75 residues. These segments are found at the periphery of the monomer and make extensive inter-subunit contacts which are responsible for the very stable octamer. A short surface helix forms contacts between two octamers, explaining the tendency of AOX to form crystals in the peroxisomes.

Hardly anything is known regarding the detoxification of surfactants in crop plants, although they are frequently treated with agrochemical formulations. Therefore, we studied transcriptomic changes in barley leaves induced in response to spraying leaf surfaces with two alcohol ethoxylates (AEs). As model surfactants, we selected the monodisperse tetraethylene glycol monododecyl (C12E4) ether and the polydisperse BrijL4. Barley plants were harvested 8 h after spraying with a 0.1% surfactant solution and changes in gene expression were analysed by RNA-sequencing (RNA-Seq). Gene expression was significantly altered in response to both surfactants. With BrijL4 more genes (9724) were differentially expressed compared to C12E4 (6197). Gene families showing pronounced up-regulation were cytochrome P450 enzymes, monooxygenases, ABC-transporters, acetyl- and methyl- transferases, glutathione-S-transferases and glycosyltransferases. These specific changes in gene expression and the postulated function of the corresponding enzymes allowed hypothesizing three potential metabolic pathways of AE detoxification in barley leaves. (i) Up-regulation of P450 cytochrome oxidoreductases suggested a degradation of the lipophilic alkyl residue (dodecyl chain) of the AEs by ω- and β- oxidation. (ii) Alternatively, the polar PEG-chain of AEs could be degraded. (iii) Instead of surfactant degradation, a further pathway of detoxification could be the sequestration of AEs into the vacuole or the apoplast (cell wall). Thus, our results show that AEs lead to pronounced changes in the expression of genes coding for proteins potentially being involved in the detoxification of surfactants.

The CAZy auxiliary activity family 3 (AA3) comprises enzymes from the glucose-methanol-choline (GMC) family of oxidoreductases, which assist the activity of other AA family enzymes via their reaction products or support the action of glycoside hydrolases in lignocellulose degradation. The AA3 family is further divided into four subfamilies, which include cellobiose dehydrogenase, glucose oxidoreductases, aryl-alcohol oxidase, alcohol (methanol) oxidase, and pyranose oxidoreductases. These different enzymes catalyze a wide variety of redox reactions with respect to substrates and co-substrates. The common feature of AA3 family members is the formation of key metabolites such as H2O2 or hydroquinones, which are required by other AA enzymes. The multiplicity of enzymatic functions in the AA3 family is reflected by the multigenicity of AA3 genes in fungi, which also depends on their lifestyle. We provide an overview of the phylogenetic, molecular, and catalytic properties of AA3 enzymes and discuss their interactions with other carbohydrate-active enzymes.

(S)-carbonyl reductase II (SCRII) from Candida parapsilosis is a short-chain alcohol dehydrogenase/reductase. It catalyses the conversion of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol with low efficiency. Sortase was reported as a molecular "stapler" for site-specific protein conjugation to strengthen or add protein functionality. Here, we describe Staphylococcus aureus sortase A-mediated crosslinking of SCRII to produce stable catalysts for efficient biotransformation. Via a native N-terminal glycine and an added GGGGSLPETGG peptide at C-terminus of SCRII, SCRII subunits were conjugated by sortase A to form crosslinked SCRII, mainly dimers and trimers. The crosslinked SCRII showed over 6-fold and 4-fold increases, respectively, in activity and k cat/K m values toward 2-hydroxyacetophenone compared with wild-type SCRII. Moreover, crosslinked SCRII was much more thermostable with its denaturation temperature (Tm) increased to 60 °C. Biotransformation result showed that crosslinked SCRII gave a product optical purity of 100% and a yield of >99.9% within 3 h, a 16-fold decrease in transformation duration with respect to Escherichia coli/pET-SCRII. Sortase A-catalysed ligation also obviously improved Tms and product yields of eight other short-chain alcohol dehydrogenases/reductases. This work demonstrates a generic technology to improve enzyme function and thermostability through sortase A-mediated crosslinking of oxidoreductases.

Decaprenylphosphoryl-β-D-ribose 2'-epimerase (DprE1) is an essential enzyme in the biosynthesis of cell wall components and a target for development of anti-tuberculosis drugs. We determined the crystal structure of a truncated form of DprE1 from Mycobacterium smegmatis in two crystal forms to up to 2.35 Å resolution. The structure extends from residue 75 to the C-terminus and shares homology with FAD-dependent oxidoreductases of the vanillyl-alcohol oxidase family including the DprE1 homologue from M. tuberculosis. The M. smegmatis DprE1 structure reported here provides further insights into the active site geometry of this tuberculosis drug target.

Streptoseomycin (STM, 1) is a bacterial macrolactone that has a unique 5/14/10/6/6-pentacyclic ring with an ether bridge. We have previously identified the biosynthetic gene cluster for 1 and characterized StmD as [6 + 4]- and [4 + 2]-bispericyclase that catalyze a reaction leading to both 6/10/6- and 10/6/6-tricyclic adducts (6 and 7). The remaining steps, especially how to install and stabilize the required 10/6/6-tricyclic core for downstream modifications, remain unknown. In this work, we have identified three oxidoreductases that fix the required 10/6/6-tryciclic core. A pair of flavin-dependent oxidoreductases, StmO1 and StmO2, catalyze the direct hydroxylation at [6 + 4]-adduct (6). Subsequently, a spontaneous [3,3]-Cope rearrangement and an enol-ketone tautomerization result in the formation of 10/6/6-tricyclic intermediate 12b, which can be further converted to a stable 10/6/6-tricyclic alcohol 11 through a ketoreduction by StmK. Crystal structure of the heterodimeric complex NtfO1-NtfO2, homologues of StmO1-StmO2 with equivalent function, reveals protein-protein interactions. Our results demonstrate that the [6 + 4]-adduct instead of [4 + 2]-adduct is the bona fide biosynthetic intermediate.

Plant genomes contain a large number of genes encoding for berberine bridge enzyme (BBE)-like enzymes. Despite the widespread occurrence and abundance of this protein family in the plant kingdom, the biochemical function remains largely unexplored. In this study, we have expressed two members of the BBE-like enzyme family from Arabidopsis thaliana in the host organism Komagataella pastoris. The two proteins, termed AtBBE-like 13 and AtBBE-like 15, were purified, and their catalytic properties were determined. In addition, AtBBE-like 15 was crystallized and structurally characterized by x-ray crystallography. Here, we show that the enzymes catalyze the oxidation of aromatic allylic alcohols, such as coumaryl, sinapyl, and coniferyl alcohol, to the corresponding aldehydes and that AtBBE-like 15 adopts the same fold as vanillyl alcohol oxidase as reported previously for berberine bridge enzyme and other FAD-dependent oxidoreductases. Further analysis of the substrate range identified coniferin, the glycosylated storage form of coniferyl alcohol, as a substrate of the enzymes, whereas other glycosylated monolignols were rather poor substrates. A detailed analysis of the motifs present in the active sites of the BBE-like enzymes in A. thaliana suggested that 14 out of 28 members of the family might catalyze similar reactions. Based on these findings, we propose a novel role of BBE-like enzymes in monolignol metabolism that was previously not recognized for this enzyme family.

Enzymatic oxidation of 5-hydroxymethylfurfural (HMF) and its oxidized derivatives was studied using three fungal enzymes: wild-type aryl alcohol oxidase (AAO) from three fungal species, wild-type peroxygenase from Agrocybe aegerita (AaeUPO), and recombinant galactose oxidase (GAO). The effect of pH on different reaction steps was evaluated and apparent kinetic data (Michaelis-Menten constants, turnover numbers, specific constants) were calculated for different enzyme-substrate ratios and enzyme combinations. Finally, the target product, 2,5-furandicarboxylic acid (FDCA), was prepared in a multi-enzyme cascade reaction combining three fungal oxidoreductases at micro-scale. Furthermore, an oxidase-like reaction is proposed for heme-containing peroxidases, such as UPO, horseradish peroxidase, or catalase, causing the conversion of 5-formyl-2-furancarboxylic acid into FDCA in the absence of exogenous hydrogen peroxide.

The enzymatic conversion of plant biomass has been recently revolutionized by the discovery of lytic polysaccharide monooxygenases (LPMOs) that carry out oxidative cleavage of polysaccharides. These very powerful enzymes are abundant in fungal saprotrophs. LPMOs require activation by electrons that can be provided by cellobiose dehydrogenases (CDHs), but as some fungi lack CDH-encoding genes, other recycling enzymes must exist. We investigated the ability of AA3_2 flavoenzymes secreted under lignocellulolytic conditions to trigger oxidative cellulose degradation by AA9 LPMOs. Among the flavoenzymes tested, we show that glucose dehydrogenase and aryl-alcohol quinone oxidoreductases are catalytically efficient electron donors for LPMOs. These single-domain flavoenzymes display redox potentials compatible with electron transfer between partners. Our findings extend the array of enzymes which regulate the oxidative degradation of cellulose by lignocellulolytic fungi.

The aim of this study was to evaluate the bioremoval mechanism of anthracycline antibiotics by the white-rot fungus B. adusta CCBAS 930. The activity of oxidoreductases and levels of phenolic compounds and free radicals were determined during the biotransformation of anthraquinone antibiotics: daunomycin (DNR) and doxorubicin (DOX) by B. adusta strain CCBAS 930. Moreover, phytotoxicity (Lepidium sativum L.), ecotoxicity (Vibrio fischeri), genotoxicity and cytotoxicity of anthraquinone dyes were evaluated before and after biological treatment. More than 80% and 90% of DNR and DOX were removed by biodegradation (decolorization). Initial solutions of DNR and DOX were characterized by eco-, phyto-, geno- and cytotoxicity. Despite efficient decolorization, secondary metabolites, toxic to bacteria, formed during biotransformation of anthracycline antibiotics in B. adusta CCBAS 930 cultures. DNR and DOX metabolites did not increase reactive oxygen species (ROS) production in human fibroblasts and resazurin reduction. DNR metabolites did not change caspase-3 activity.

Microbial degradation of lignin and its related aromatic compounds has great potential for the sustainable production of chemicals and bioremediation of contaminated soils. We previously isolated Pseudomonas sp. strain 9.1 from historical waste deposits (forming so-called fiber banks) released from pulp and paper mills along the Baltic Sea coast. The strain accumulated vanillyl alcohol during growth on vanillin, and while reported in other microbes, this phenotype is less common in wild-type pseudomonads. As the reduction of vanillin to vanillyl alcohol is an undesired trait in Pseudomonas strains engineered to accumulate vanillin, connecting the strain 9.1 phenotype with a genotype would increase the fundamental understanding and genetic engineering potential of microbial vanillin metabolism. The genome of Pseudomonas sp. 9.1 was sequenced and assembled. Annotation identified oxidoreductases with homology to Saccharomyces cerevisiae alcohol dehydrogenase ScADH6p, known to reduce vanillin to vanillyl alcohol, in both the 9.1 genome and the model strain Pseudomonas putida KT2440. Recombinant expression of the Pseudomonas sp. 9.1 FEZ21_09870 and P. putida KT2440 PP_2426 (calA) genes in Escherichia coli revealed that these open reading frames encode aldehyde reductases that convert vanillin to vanillyl alcohol, and that P. putida KT2440 PP_3839 encodes a coniferyl alcohol dehydrogenase that oxidizes coniferyl alcohol to coniferyl aldehyde (i.e., the function previously assigned to calA). The deletion of PP_2426 in P. putida GN442 engineered to accumulate vanillin resulted in a decrease in by-product (vanillyl alcohol) yield from 17% to ∼1%. Based on these results, we propose the reannotation of PP_2426 and FEZ21_09870 as areA and PP_3839 as calA-IIIMPORTANCE Valorization of lignocellulose (nonedible plant matter) is of key interest for the sustainable production of chemicals from renewable resources. Lignin, one of the main constituents of lignocellulose, is a heterogeneous aromatic biopolymer that can be chemically depolymerized into a heterogeneous mixture of aromatic building blocks; those can be further converted by certain microbes into value-added aromatic chemicals, e.g., the flavoring agent vanillin. We previously isolated a Pseudomonas sp. strain with the (for the genus) unusual trait of vanillyl alcohol production during growth on vanillin. Whole-genome sequencing of the isolate led to the identification of a vanillin reductase candidate gene whose deletion in a recombinant vanillin-accumulating P. putida strain almost completely alleviated the undesired vanillyl alcohol by-product yield. These results represent an important step toward biotechnological production of vanillin from lignin using bacterial cell factories.

Aldehyde:ferredoxin oxidoreductases (AORs) have been isolated and biochemically-characterized from a handful of anaerobic or facultative aerobic archaea and bacteria. They catalyze the ferredoxin (Fd)-dependent oxidation of aldehydes to acids. Recently, the involvement of AOR in the reduction of organic acids to alcohols with electrons derived from sugar or synthesis gas was demonstrated, with alcohol dehydrogenases (ADHs) carrying out the reduction of the aldehyde to the alcohol (AOR-ADH pathway). Here, we describe the biochemical characterization of an AOR of the thermophilic fermentative bacterium Thermoanaerobacter sp. strain X514 (AORX514). The putative aor gene (Teth514_1380) including a 6x-His-tag was introduced into the genome of the genetically-accessible, related species Thermoanaerobacter kivui. The protein was purified to apparent homogeneity, and indeed revealed AOR activity, as measured by acetaldehyde-dependent ferredoxin reduction. AORX514 was active over a wide temperature (10 to 95 °C) and pH (5.5 to 11.5) range, utilized a wide variety of aldehydes (short and branched-chained, aliphatic, aromatic) and resembles archaeal sensu stricto AORs, as the protein is active in a homodimeric form. The successful, recombinant production of AORX514 in a related, well-characterized and likewise strict anaerobe paves the road towards structure-function analyses of this enzyme and possibly similar oxygen-sensitive or W/Mo-dependent proteins in the future.

Clostridium formicoaceticum, a Gram-negative mixotrophic homoacetogen, produces acetic acid as the sole metabolic product from various carbon sources, including fructose, glycerol, formate, and CO2. Its genome of 4.59-Mbp contains a highly conserved Wood-Ljungdahl pathway gene cluster with the same layout as that in other mixotrophic acetogens, including Clostridium aceticum, Clostridium carboxidivorans, and Clostridium ljungdahlii. For energy conservation, C. formicoaceticum does not have all the genes required for the synthesis of cytochrome or quinone used for generating proton gradient in H+-dependent acetogens such as Moorella thermoacetica; instead, it has the Rnf system and a Na+-translocating ATPase similar to the one in Acetobacterium woodii. Its growth in both heterotrophic and autotrophic media were dependent on the sodium concentration. C. formicoaceticum has genes encoding acetaldehyde dehydrogenases, alcohol dehydrogenases, and aldehyde oxidoreductases, which could convert acetyl-CoA and acetate to ethanol and butyrate to butanol under excessive reducing equivalent conditions.

The basidiomycete Chondrostereum purpureum (Silverleaf fungus) is a saprotroph and plant pathogen commercially used for combatting forest "weed" trees in vegetation management. However, little is known about its lignocellulose-degrading capabilities and the enzymatic machinery that is responsible for the degradative potential, and it is not yet clear to which group of wood-rot fungi it actually belongs. Here, we sequenced and analyzed the draft genome of C. purpureum (41.2 Mbp) and performed a quantitative proteomic approach during growth in submerged and solid-state cultures based on soybean meal suspension or containing beech wood supplemented with phenol-rich olive mill residues, respectively. The fungus harbors characteristic lignocellulolytic hydrolases (GH6 and GH7) and oxidoreductases (e.g. laccase, heme peroxidases). High abundance of some of these genes (e.g. 45 laccases, nine GH7) can be explained by gene expansion, e.g. identified for the laccase orthogroup ORTHOMCL11 that exhibits a total of 18 lineage-specific duplications. Other expanded genes families encode for proteins more related to a pathogenic lifestyle (e.g. protease and cytochrome P450s). The fungus responds to the presence of complex growth substrates (lignocellulose, phenolic residues) by the secretion of most of these lignocellulolytic and lignin-modifying enzymes (e.g. alcohol and aryl alcohol oxidases, laccases, GH6, GH7). Based on the genetic and enzymatic constitution, we consider the 'marasmioid' fungus C. purpureum as a 'phytopathogenic' white-rot fungus (WRF) that possesses a complex extracellular enzyme machinery to accomplish efficient lignocellulose degradation during both saprotrophic and phytopathogenic life phases.

Synthesis gas (syngas) fermentation by anaerobic acetogenic bacteria employing the Wood-Ljungdahl pathway is a bioprocess for production of biofuels and biocommodities. The major fermentation products of the most relevant biocatalytic strains (Clostridium ljungdahlii, C. autoethanogenum, C. ragsdalei, and C. coskatii) are acetic acid and ethanol. A comparative metabolic and genomic analysis using the mentioned biocatalysts might offer targets for metabolic engineering and thus improve the production of compounds apart from ethanol. Autotrophic growth and product formation of the four wild type (WT) strains were compared in uncontrolled batch experiments. The genomes of C. ragsdalei and C. coskatii were sequenced and the genome sequences of all four biocatalytic strains analyzed in comparative manner. Growth and product spectra (acetate, ethanol, 2,3-butanediol) of C. autoethanogenum, C. ljungdahlii, and C. ragsdalei were rather similar. In contrast, C. coskatii produced significantly less ethanol and its genome sequence lacks two genes encoding aldehyde:ferredoxin oxidoreductases (AOR). Comparative genome sequence analysis of the four WT strains revealed high average nucleotide identity (ANI) of C. ljungdahlii and C. autoethanogenum (99.3%) and C. coskatii (98.3%). In contrast, C. ljungdahlii WT and C. ragsdalei WT showed an ANI-based similarity of only 95.8%. Additionally, recombinant C. ljungdahlii strains were constructed that harbor an artificial acetone synthesis operon (ASO) consisting of the following genes: adc, ctfA, ctfB, and thlA (encoding acetoacetate decarboxylase, acetoacetyl-CoA:acetate/butyrate:CoA-transferase subunits A and B, and thiolase) under the control of thlA promoter (P thlA ) from C. acetobutylicum or native pta-ack promoter (P pta-ack ) from C. ljungdahlii. Respective recombinant strains produced 2-propanol rather than acetone, due to the presence of a NADPH-dependent primary-secondary alcohol dehydrogenase that converts acetone to 2-propanol. Furthermore, the ClosTron(TM) system was used to construct an adhE1 integration mutant. These results provide extensive insights into genetic features of industrially relevant bacterial biocatalysts and expand the toolbox for metabolic engineering of acetogenic bacteria able to ferment syngas.

Saccharomyces cerevisiae plays an important role in the heterologous expression of an array of proteins due to its easy manipulation, low requirements and ability for protein post-translational modifications. The implementation of the preproleader secretion signal of the α-factor mating pheromone from this yeast contributes to increase the production yields by targeting the foreign protein to the extracellular environment. The use of this signal peptide combined with enzyme-directed evolution allowed us to achieve the otherwise difficult functional expression of fungal laccases in S. cerevisiae, obtaining different evolved α-factor preproleader sequences that enhance laccase secretion. However, the design of a universal signal peptide to enhance the production of heterologous proteins in S. cerevisiae is a pending challenge. We describe here the optimisation of the α-factor preproleader to improve recombinant enzyme production in S. cerevisiae through two parallel engineering strategies: a bottom-up design over the native α-factor preproleader (αnat) and a top-down design over the fittest evolved signal peptide obtained in our lab (α9H2 leader). The goal was to analyse the effect of mutations accumulated in the signal sequence throughout iterations of directed evolution, or of other reported mutations, and their possible epistatic interactions. Both approaches agreed in the positive synergism of four mutations (Aα9D, Aα20T, Lα42S, Dα83E) contained in the final optimised leader (αOPT), which notably enhanced the secretion of several fungal oxidoreductases and hydrolases. Additionally, we suggest a guideline to further drive the heterologous production of a particular enzyme based on combinatorial saturation mutagenesis of positions 86th and 87th of the αOPT leader fused to the target protein.

The application of biocatalysis for the asymmetric reduction of activated C=C is a powerful tool for the manufacture of high-value chemical commodities. The biocatalytic potential of "-ene" reductases from the Old Yellow Enzyme (OYE) family of oxidoreductases is well-known; however, the specificity of these enzymes toward mainly small molecule substrates has highlighted the need to discover "-ene" reductases from different enzymatic classes to broaden industrial applicability. Here, we describe the characterization of a flavin-free double bond reductase from Nicotiana tabacum (NtDBR), which belongs to the leukotriene B4 dehydrogenase (LTD) subfamily of the zinc-independent, medium chain dehydrogenase/reductase superfamily of enzymes. Using steady-state kinetics and biotransformation reactions, we have demonstrated the regio- and stereospecificity of NtDBR against a variety of α,β-unsaturated activated alkenes. In addition to catalyzing the reduction of typical LTD substrates and several classical OYE-like substrates, NtDBR also exhibited complementary activity by reducing non-OYE substrates (i.e., reducing the exocyclic C=C double bond of (R)-pulegone) and in some cases showing an opposite stereopreference in comparison with the OYE family member pentaerythritol tetranitrate (PETN) reductase. This serves to augment classical OYE "-ene" reductase activity and, coupled with its aerobic stability, emphasizes the potential industrial value of NtDBR. Furthermore, we also report the X-ray crystal structures of the holo-, binary NADP(H)-bound, and ternary [NADP+ and 4-hydroxy-3-methoxycinnamaldehyde (9a)-bound] NtDBR complexes. These will underpin structure-driven site-saturated mutagenesis studies aimed at enhancing the reactivity, stereochemistry, and specificity of this enzyme.