Kemp Elimination Catalyzed by Naturally Occurring Aldoxime Dehydratases

Recently, the Kemp elimination reaction has been extensively studied in computational enzyme design of new catalysts, as no natural enzyme has evolved to catalyze this reaction. In contrast to in silico enzyme design, we were interested in searching for Kemp eliminase activity in natural enzymes with catalytic promiscuity. Based on similarities of substrate structures and reaction mechanisms, we assumed that the active sites of naturally abundant aldoxime dehydratases have the potential to catalyze the non‐natural Kemp elimination reaction. We found several aldoxime dehydratases that are efficient catalysts of this reaction. Although a few natural enzymes have been identified with promiscuous Kemp eliminase activity, to the best of our knowledge, this is a rare example of Kemp elimination catalyzed by naturally occurring enzymes with high catalytic efficiency.     

Ultrahigh‐Throughput FACS‐Based Screening for Directed Enzyme Evolution

Directed enzyme evolution has proven to be a powerful tool for improving a range of properties of enzymes through consecutive rounds of diversification and selection. However, its success depends heavily on the efficiency of the screening strategy employed. Fluorescence‐activated cell sorting (FACS) has recently emerged as a powerful tool for screening enzyme libraries due to its high sensitivity and its ability to analyze as many as 10⁸ mutants per day. Applications of FACS screening have allowed the isolation of enzyme variants with significantly improved activities, altered substrate specificities, or even novel functions. This review discusses FACS‐based screening for enzymatic activity and its potential application for the directed evolution of enzymes, ribozymes, and catalytic antibodies.     

Enhancing the Efficiency and Regioselectivity of P450 Oxidation Catalysts by Unnatural Amino Acid Mutagenesis

The development of effective strategies for modulating the reactivity and selectivity of cytochrome P450 enzymes represents a key step toward expediting the use of these biocatalysts for synthetic applications. We have investigated the potential of unnatural amino acid mutagenesis to aid efforts in this direction. Four unnatural amino acids with diverse aromatic side chains were incorporated at 11 active‐site positions of a substrate‐promiscuous CYP102A1 variant. The resulting “uP450s” were then tested for their catalytic activity and regioselectivity in the oxidation of two representative substrates: a small‐molecule drug and a natural product. Large shifts in regioselectivity resulted from these single mutations, and in particular, for para‐acetyl‐Phe substitutions at positions close to the heme cofactor. Screening this mini library of uP450s enabled us to identify P450 catalysts for the selective hydroxylation of four aliphatic positions in the target substrates, including a C(sp³)?H site not oxidized by the parent enzyme. Furthermore, we discovered a general activity‐enhancing effect of active‐site substitutions involving the unnatural amino acid para‐amino‐Phe, which resulted in P450 catalysts capable of supporting the highest total turnover number reported to date on a complex molecule (34 650). The functional changes induced by the unnatural amino acids could not be reproduced by any of the 20 natural amino acids. This study thus demonstrates that unnatural amino acid mutagenesis constitutes a promising new strategy for improving the catalytic activity and regioselectivity of P450 oxidation catalysts.     

Analysis of the Catalytic Mechanism of Bifunctional Triterpene/Sesquarterpene Cyclase: Tyr167 Functions To Terminate Cyclization of Squalene at the Bicyclic Step

Onoceroids are a group of triterpenes biosynthesized from squalene or dioxidosqualene by cyclization from both termini. We previously identified a bifunctional triterpene/sesquarterpene cyclase (TC) that constructs a tetracyclic scaffold from tetraprenyl‐β‐curcumene (C₃₅) but a bicyclic scaffold from squalene (C₃₀) in the first reaction. TC also accepts the bicyclic intermediate as a substrate and generates tetracyclic and pentacyclic onoceroids in the second reaction. In this study, we analyzed the catalytic mechanism of an onoceroid synthase by using mutated enzymes. TC^Y167A produced an unnatural tricyclic triterpenol, but TC^Y167L, TC^Y167F, and TC^Y167W formed small quantities of tricyclic compounds, which suggested that the bulk size at Y167 contributed to termination of the cyclization of squalene at the bicyclic step. Our findings provide insight into the unique catalytic mechanism of TC, which triggers different cyclization modes depending on the substrate. These findings may facilitate the large‐scale production of an onoceroid for which natural sources are limited.     

Evaluating the Catalytic Potential of a General RNA-Cleaving FANA Enzyme

The discovery of synthetic genetic polymers (XNAs) with catalytic activity demonstrates that natural genetic polymers are not unique in their ability to function as enzymes. However, all known examples of in vitro selected XNA enzymes function with lower activity than their natural counterparts, suggesting that XNAs might be limited in their ability to fold into structures with high catalytic activity. To explore this problem, we evaluated the catalytic potential of FANAzyme 12–7, an RNA-cleaving catalyst composed entirely of 2′-fluoroarabino nucleic acid (FANA) that was evolved to cleave RNA at a specific phosphodiester bond located between an unpaired guanine and a paired uracil in the substrate recognition arm. Here, we show that this activity extends to chimeric DNA substrates that contain a central riboguanosine (riboG) residue at the cleavage site. Surprisingly, FANAzyme 12–7 rivals known DNAzymes that were previously evolved to cleave chimeric DNA substrates under physiological conditions. These data provide convincing evidence that FANAzyme 12–7 maintains the catalytic potential of equivalent DNAzymes, which has important implications for the evolution of XNA catalysts and their contributions to future applications in synthetic biology.     

Molecular basis of lipase stereoselectivity

Lipases exhibit specific catalytic properties that make them attractive to biotechnological applications. Most important are the broad substrate specificity and the regio‐ and stereoselectivity of lipases. Despite mechanistic and structural similarities lipases differ significantly with respect to stereoselectivity toward natural and synthetic substrates. Models developed to describe and predict stereoselectivity toward certain types of synthetic substrates, e. g., secondary alcohols cannot be applied to natural acylglycerols, that are hydrolyzed by several animal and microbial lipases in a regioselective or stereoselective manner. Therefore, computer‐aided molecular modeling studies were used in order to predict the stereopreference of lipases toward triradylglycerols. Lipase variants with modified stereoselectivity properties toward triacylglycerols were engineered by re‐designing the recombinant enzyme. To understand the interactions governing lipase stereoselectivity towards natural substrates, knowledge of the structure of enzyme‐substrate complexes at the atomic level is essential. Such information can be obtained by X‐ray or NMR analysis of covalent enzyme‐inhibitor complexes. The crystal structures of enzymes complexed with triacylglycerol analog inhibitors allowed the identification of distinct binding sites for the three hydrophobic chains of the inhibitor.     

Engineering of a Cytidine 5′‐Monophosphate‐Sialic Acid Synthetase for Improved Tolerance to Functional Sialic Acids

Sialic acid‐containing glycoconjugates at the cell surface are of high importance in carbohydrate‐mediated recognition phenomena in physiological and pathological events, as well as in bacterial or viral infection. A key step in the enzymatic synthesis of natural sialoconjugates and functional synthetic analogues is the activation of sialic acids to cytidine 5′‐monophosphate (CMP)‐sialic acid intermediates catalyzed by CMP‐sialic acid synthetase (CSS). Based on our recently developed aligned protein model of substrate binding and a simple colorimetric screening assay, we have engineered the CSS from Neisseria meningitidis by structure‐guided site‐specific saturation mutagenesis at positions 192/193 to generate enzymes with broadened substrate scope. Top hits, including the F192S/F193Y variant, display an improvement of up to 70‐fold catalytic efficiency relative to wild‐type CSS for the conversion of sterically demanding N‐acyl modified sialic acid analogues, without compromising protein stability. Such significantly enhanced substrate capacity is a major step forward to realizing a generalized chemo‐enzymatic strategy for the efficient preparation of neo‐sialoconjugate libraries, demonstrated by the highly efficient, regio‐ and stereospecific synthesis of 2,6‐sialyllactose analogues by enzymatic coupling to the highly substrate tolerant α2,6‐sialyltransferase from Photobacterium leiognathi JT‐SHIZ‐145. Our results further document the unusual versatility of the N. meningitidis CSS and engineered variants for a common synthetic approach to sialoconjugates comprising a large diversity of natural and non‐natural sialic acid forms without the need for post‐synthetic enzymatic modification.

     

Expanding the Scope of Sortase‐Mediated Ligations by Using Sortase Homologues

Sortase‐catalyzed transacylation reactions are widely used for the construction of non‐natural protein derivatives. However, the most commonly used enzyme for these strategies (sortase A from Staphylococcus aureus) is limited by its narrow substrate scope. To expand the range of substrates compatible with sortase‐mediated reactions, we characterized the in vitro substrate preferences of eight sortase A homologues. From these studies, we identified sortase A enzymes that recognize multiple substrates that are unreactive toward sortase A from S. aureus. We further exploited the ability of sortase A from Streptococcus pneumoniae to recognize an LPATS substrate to perform a site‐specific modification of the N‐terminal serine residue in the naturally occurring antimicrobial peptide DCD‐1L. Finally, we unexpectedly observed that certain substrates (LPATXG, X=Nle, Leu, Phe, Tyr) were susceptible to transacylation at alternative sites within the substrate motif, and sortase A from S. pneumoniae was capable of forming oligomers. Overall, this work provides a foundation for the further development of sortase enzymes for use in protein modification.     

Single‐Turnover Kinetics Reveal a Distinct Mode of Thiamine Diphosphate‐Dependent Catalysis in Vitamin K Biosynthesis

MenD, or (1R,2S,5S,6S)‐2‐succinyl‐5‐enolpyruvyl‐6‐hydroxycyclohex‐3‐ene‐1‐carboxylate (SEPHCHC) synthase, uses a thiamine diphosphate (ThDP)‐dependent tetrahedral Breslow intermediate rather than a canonical enamine for catalysis in the biosynthesis of vitamin K. By real‐time monitoring of the cofactor chemical state with circular dichroism spectroscopy, we found that a new post‐decarboxylation intermediate was formed from a multistep process that was rate limited by binding of the α‐ketoglutarate substrate before it quickly relaxed to the characterized tetrahedral Breslow intermediate. In addition, the chemical steps leading to the reactive post‐decarboxylation intermediates were not affected by the electrophilic substrate, isochorismate, whereas release of the product was found to limit the whole catalytic process. Moreover, these intermediates are likely kinetically stabilized owing to the low biological availability of isochorismate under physiological conditions, in contrast to the tight coupling of enamine formation with binding of the electrophilic acceptor in some other ThDP‐dependent enzymes. Together with the unusual tetrahedral structure of the intermediates, these findings strongly support a new ThDP‐dependent catalytic mode distinct from canonical enamine chemistry.     

Mathematical modeling of water uptake through diffusion in 3D inhomogeneous swelling substrates

Diffusion‐driven water uptake in a substrate (imbibition) is a subject of great interest in the field of food technology. This is a particular challenge for rice grains that are preprocessed to accelerate the water uptake, i.e., to reduce the cooking time. Rice preprocessing disrupts the mesostructural order of starch and induces a microporous structure in the grains. The meso‐ and microstructural length scales have not been considered in joint approach until now. The (re)hydration of rice grains can be modeled by free (concentration‐driven) diffusion or by water demand‐driven diffusion. The latter is driven by the ceiling moisture content related to the extent of gelatinization of the rice substrate network. This network can be regarded as a fractal structure. As the spatial resolution of our models is limited, we choose to model the apparent water transport by a set of coupled partial differential equations (PDEs). Current models of water uptake are often limited to a single dimension, and the swelling of the substrate is not taken into account. In this article, we derive a set of PDEs to model water uptake in a three‐dimensional (3D) inhomogeneous substrate for different types of water diffusion as well as the swelling of the substrate during water uptake. We will present simulation results for different 3D (macroscopic) structures and diffusion models and compare these results, qualitatively, with the experimental results acquired from magnetic resonance imaging. © 2009 American Institute of Chemical Engineers AIChE J, 2009     

C?C Hydrolases for Biocatalysis

Although C C bond hydrolases are distributed widely in Nature, they has as yet have received only limited attention in the area of biocatalysis compared to their counterpart the C‐heteroatom hydrolases, such as lipases and proteases. However, the substrate range of C C hydrolases, and their non‐dependence on cofactors, suggest that these enzymes may have considerable potential for applications in synthesis. In addition, hydrolases such as the β‐diketone hydrolase from Rhodococcus (OCH) are known, that catalyse the formation of interesting chiral intermediates. Further enzymes, such as kynureninase and a meta‐cleavage product hydrolase (MhpC), are able to catalyse carbon‐carbon bond formation, suggesting wider applications in biocatalysis than previously envisaged. In this review, the distribution, catalytic characteristics and applications of C C hydrolases are described, with a view to assessing their potentialfor use in biocatalytic processes in the future.     

Fusion Enzymes Involved in Biosynthetic Tailoring Reactions in Fungi

Enzyme fusion, the fusion of enzymes with different domains to a single protein, has been widely recognized as a promising strategy in the development of biocatalysts. Nature has evolved gene fusion to combine different catalytic enzymes to function as a fusion enzyme, and this strategy is utilized in many natural product biosynthetic pathways. Owing to rapid advances in genome sequencing and biosynthetic pathway characterization, there is increasing interest in fusion enzymes from fungal biosynthetic pathways, particularly those involved in tailoring steps. This concept aims to provide an up-to-date overview of fusion enzymes that catalyze tailoring reactions in the biosynthesis of fungal secondary metabolites. Since fungal fusion enzymes are often associated with novel metabolites, this pioneering work may stimulate the exploration of the structural diversity of fungal natural products through genome mining of the untapped biosynthetic pathways involving fusion enzymes.     

Enhancement of Candida parapsilosis catalyzing deracemization of (R,S)‐1‐phenyl‐1, 2‐ethanediol: agitation speed control during cell cultivation

BACKGROUND: Microbial cells have been used widely in biosynthesis of chiral alcohols. However, research concerning the effect of oxygen supply in cultivation on biocatalytic activity of whole cells in organic synthesis is limited. This study improved the reaction efficiency of Candida parapsilosis catalyzing (R,S)‐1‐phenyl‐1,2‐ethanediol (PED) deracemization by controlling agitation during cell cultivation. RESULTS: The increase of dissolved oxygen concentration by adjusting agitation speed from 200 to 300 rpm at aeration rate 1.5 vvm significantly improved the cell growth of C. parapsilosis and the activities of two key enzymes involved in deracemization. (S)‐PED with higher optical purity of 98.23%e.e. and yield of 82.94% was formed. Compared with the initial fermentation conditions at aeration rate 0.75 vvm and agitation speed 200 rpm, enhanced oxygen supply conditions afforded better cells for highly efficient conversion under higher substrate concentration. CONCLUSION: Oxygen supply had a significant effect on cell growth and catalytic activity of C. parapsilosis catalyzing asymmetric oxidoreduction. By conveniently controlling agitation in cultivation, cell activity and key enzymes production for a complex reaction of concurrent tandem oxidation and reduction processes can easily be conducted, which could help to cultivate cells catalyzing synthesis of interested chiral compounds. Copyright © 2008 Society of Chemical Industry     

Regio‐ and Stereospecific Prenylation of Flavonoids by Sophora flavescens Prenyltransferase

Prenylflavonoids are valuable natural products that are widely distributed in plants. They often possess divergent biological properties, including phytoestrogenic, anti‐bacterial, anti‐tumor, and anti‐diabetic activities. The reaction catalyzed by prenyltransferases represents a Friedel–Crafts alkylation of the flavonoid skeleton in the biosynthesis of natural prenylflavonoids and often contributes to the structural diversity and biological activity of these compounds. However, only a few plant flavonoid prenyltransferases have been identified thus far, and these prenyltransferases exhibit strict substrate specificity and low catalytic efficiency. In this article, a flavonoid prenyltransferase from Sophora flavescens, SfFPT, has been identified that displays high catalytic efficiency with high regiospecificity acting on C‐8 of structurally different types of flavonoid (i.e., flavanone, flavone, flavanonol, and dihydrochalcone, etc.). Furthermore, SfPFT exhibits strict stereospecificity for levorotatory flavanones to produce (2S)‐prenylflavanones. This study is the first to demonstrate the substrate promiscuity and stereospecificity of a plant flavonoid prenyltransferase in vitro. Given its substrate promiscuity and high catalytic efficiency, SfFPT can be used as an environmentally friendly and efficient biological catalyst for the regio‐ and stereospecific prenylation of flavonoids to produce bioactive compounds for potential therapeutic applications.     

Catalytic porous ceramic prepared in‐situ by sol‐gelation for butane‐to‐syngas processing in microreactors

In this study, a novel flow‐based method is presented to place catalytic nanoparticles into a reactor by sol‐gelation of a porous ceramic consisting of Rh/ceria/zirconia nanoparticles, silica sand, ceramic binder, and a gelation agent. This method allows for the placement of a liquid precursor containing the catalyst into the final reactor geometry without the need of impregnating or coating of a substrate with the catalytic material. The so generated foam‐like porous ceramic shows properties highly appropriate for use as catalytic reactor material, e.g., reasonable pressure drop due to its porosity, high thermal and catalytic stability, and excellent catalytic behavior. To investigate the catalytic activity, microreactors containing this foam‐like ceramic are employed for the production of hydrogen and carbon monoxide‐rich syngas from butane. The effect of operating parameters such as the inlet flow rate on the hydrocarbon processing is analyzed and the limitation of the reactor by diffusion mass transport is investigated. © 2009 American Institute of Chemical Engineers AIChE J, 2009     

Catalytic Mechanism of the Hotdog‐Fold Thioesterase PA1618 Revealed by X‐ray Structure Determination of a Substrate‐Bound Oxygen Ester Analogue Complex

Thioesterase activity accounts for the majority of the activities in the hotdog‐fold superfamily. The structures and mechanisms of catalysis for many hotdog enzymes have been elucidated by X‐ray crystallography and kinetics to probe the specific substrate usage and cellular functions. However, structures of hotdog thioesterases in complexes with substrate analogues reported to date utilize ligands that either represent truncations of the substrate or include additional atoms to prevent hydrolysis. Here we present the synthesis of an isosteric and isoelectronic substrate analogue—benzoyl‐OdCoA—and the X‐ray crystal structure of a complex of the analogue with Pseudomonas aeruginosa hotdog thioesterase PA1618 (at 1.72 Å resolution). The complex is compared with that of the “imperfect” substrate analogue phenacyl‐CoA, refined to a resolution of 1.62 Å. Kinetic and structural results are consistent with Glu64 as the catalytic residue and with the involvement of Gln49 in stabilization of the transition state. Structural comparison of the two ligand‐bound structures revealed a crucial ordered water molecule coordinated in the active site of the benzoyl‐OdCoA structure but not present in the phenacyl‐CoA‐bound structure. This suggests a general base mechanism of catalysis in which Glu64 activates the coordinated water nucleophile. Together, our findings reveal the importance of a closely similar substrate analogue to determine the true substrate binding and catalytic mechanism.     

Immobilization of horseradish peroxidase on chitosan for use in nonaqueous media

Chitosan, a natural polysaccharide, was used for the covalent immobilization of horseradish peroxidase, an enzyme of high synthetic utility, with the carbodiimide method. Of the enzyme, 62% was immobilized on chitosan when 1‐ethyl‐3‐(3‐dimethylaminopropyl carbodiimide) was used as the peptide coupling agent. The influence of different parameters, such as the enzyme concentration, carbodiimide concentration, and incubation period, on the activity retention of the immobilized enzyme was investigated. Kinetic studies using horseradish peroxidase immobilized on chitosan revealed the effects of several parameters, such as the substrate hydrophilicity and hydrophobicity, the solubility of substrates in the medium, the solvent hydrophobicity, and the support aquaphilicity, on the catalytic activity of the immobilized enzyme in nonaqueous media. General rules for the optimization of solvents for nonaqueous enzymology based on the partitioning of the solvent were not applicable for the immobilized horseradish peroxidase. The catalytic efficiency was greatest when o‐phenylene diamine was used as the substrate and least when guaiacol was used. The aquaphilicity of the support played an important role in the kinetics of the immobilized horseradish peroxidase in water‐miscible solvents. The results were promising for the future development of chitosan‐immobilized enzymes for use in organic media. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 88: 1456–1464, 2003     

tRNA Processing by Protein‐Only versus RNA‐Based RNase P: Kinetic Analysis Reveals Mechanistic Differences

In Arabidopsis thaliana, RNase P function, that is, endonucleolytic tRNA 5′‐end maturation, is carried out by three homologous polypeptides (“proteinaceous RNase P” (PRORP) 1, 2 and 3). Here we present the first kinetic analysis of these enzymes. For PRORP1, a specificity constant (k_react/K_m(sto)) of 3×10⁶ M ^?1 min^?1 was determined under single‐turnover conditions. We demonstrate a fundamentally different sensitivity of PRORP enzymes to an Rp‐phosphorothioate modification at the canonical cleavage site in a 5′‐precursor tRNA substrate; whereas processing by bacterial RNase P is inhibited by three orders of magnitude in the presence of this sulfur substitution and Mg²⁺ as the metal‐ion cofactor, the PRORP enzymes are affected by not more than a factor of five under the same conditions, without significantly increased miscleavage. These findings indicate that the catalytic mechanism utilized by proteinaceous RNase P is different from that of RNA‐based bacterial RNase P, taking place without a direct metal‐ion coordination to the (pro‐)Rp substituent. As Rp‐phosphorothioate and inosine modification at all 26 G residues of the tRNA body had only minor effects on processing by PRORP, we conclude that productive PRORP–substrate interaction is not critically dependent on any of the affected (pro‐)Rp oxygens or guanosine 2‐amino groups.     

In Vitro Reconstitution of Formylglycine‐Generating Enzymes Requires Copper(I)

Formylglycine‐generating enzymes (FGEs) catalyze O₂‐dependent conversion of specific cysteine residues of arylsulfatases and alkaline phosphatases into formylglycine. The ability also to introduce unique aldehyde functions into recombinant proteins makes FGEs a powerful tool for protein engineering. One limitation of this technology is poor in vitro activity of reconstituted FGEs. Although FGEs have been characterized as cofactor‐free enzymes we report that the addition of one equivalent of Cu^I increases catalytic efficiency more than 20‐fold and enables the identification of stereoselective C?H bond cleavage at the substrate as the rate‐limiting step. These findings remove previous limitations of FGE‐based protein engineering and also pose new questions about the catalytic mechanism of this O₂‐utilizing enzyme.