Structure-based design,synthesis of novel probes for cytochrome P450 OleT

Cytochrome P450 OleT_SA, a new cytochrome P450 enzyme from Staphylococcus aureus, catalyzes the oxidative decarboxylation and hydroxylation of fatty acids to generate terminal alkenes and fatty alcohols. The mechanism of this bifurcative chemistry remains largely unknown. Herein, a class of derivatized fatty acids were synthesized as probes to investigate the effects of substrate structure on the product type of P450 OleT_SA. The results demonstrate that the fine-tuned structure of substrates, even in a remote distance from the carboxyl group, significantly regulates OleT catalyzed decarboxylation/hydroxylation reactions. Molecular docking analysis indicated the potential interactions between the carboxylate groups of different probes and the enzyme active center which was attributed to the bifurcative chemistry.     

高通量筛选策略在细胞色素P450单加氧酶定向进化中的应用

细胞色素P450单加氧酶具有催化活性混杂性的特点,可以催化多种氧化反应,因而在生物催化领域受到了极大的关注。然而P450单加氧酶往往存在催化活性低、稳定性差、区域和立体选择性不理想等问题,从而限制了其在生物催化领域的广泛运用。蛋白质定向进化的发展与运用为改善P450单加氧酶的催化性能提供了有效的途径,而一种高效的高通量筛选策略是保证酶蛋白定向进化成功实施的关键。本文综述了P450单加氧酶定向进化过程中高通量筛选策略的最新进展。     

Enzymatic C4-Epimerization of UDP-Glucuronic Acid: Precisely Steered Rotation of a Transient 4-Keto Intermediate for an Inverted Reaction without Decarboxylation

UDP-glucuronic acid (UDP-GlcA) 4-epimerase illustrates an important problem regarding enzyme catalysis: balancing conformational flexibility with precise positioning. The enzyme coordinates the C4-oxidation of the substrate by NAD⁺ and rotation of a decarboxylation-prone β-keto acid intermediate in the active site, enabling stereoinverting reduction of the keto group by NADH. We reveal the elusive rotational landscape of the 4-keto intermediate. Distortion of the sugar ring into boat conformations induces torsional mobility in the enzyme's binding pocket. The rotational endpoints show that the 4-keto sugar has an undistorted ⁴C₁ chair conformation. The equatorially placed carboxylate group disfavors decarboxylation of the 4-keto sugar. Epimerase variants lead to decarboxylation upon removal of the binding interactions with the carboxylate group in the opposite rotational isomer of the substrate. Substitutions R185A/D convert the epimerase into UDP-xylose synthases that decarboxylate UDP-GlcA in stereospecific, configuration-retaining reactions.     

Screening mutant libraries of T7 RNA polymerase for candidates with increased acceptance of 2'-modified nucleotides

We present a screening assay based on fluorescence readout for the directed evolution of T7 RNA polymerase variants with acceptance of 2'-modified nucleotides. By using this screening we were able to identify a T7 RNA polymerase mutant with increased acceptance of 2'-methylseleno-2'-deoxyuridine 5'-triphosphate.     

A high-throughput screening assay for hydroxynitrile lyase activity

A high-throughput screening assay for hydroxynitrile lyase activity accepting a wide range of HNL-substrates is presented, which is useful either for enzyme fingerprinting or screening of huge variant libraries generated in metagenome or directed evolution approaches.     

Enhanced Electrosynthetic Hydrogen Evolution by Hydrogenases Embedded in a Redox-Active Hydrogel

Molecular hydrogen is a major high-energy carrier for future energy technologies, if produced from renewable electrical energy. Hydrogenase enzymes offer a pathway for bioelectrochemically producing hydrogen that is advantageous over traditional platforms for hydrogen production because of low overpotentials and ambient operating temperature and pressure. However, electron delivery from the electrode surface to the enzyme's active site is often rate-limiting. Here, it is shown that three different hydrogenases from Clostridium pasteurianum and Methanococcus maripaludis, when immobilized at a cathode in a cobaltocene-functionalized polyallylamine (Cc-PAA) redox polymer, mediate rapid and efficient hydrogen evolution. Furthermore, it is shown that Cc-PAA-mediated hydrogenases can operate at high faradaic efficiency (80–100 %) and low apparent overpotential (−0.578 to −0.593 V vs. SHE). Specific activities of these hydrogenases in the electrosynthetic Cc-PAA assay were comparable to their respective activities in traditional methyl viologen assays, indicating that Cc-PAA mediates electron transfer at high rates, to most of the embedded enzymes.     

Oxidative Decarboxylation of Short‐Chain Fatty Acids to 1‐Alkenes

The enzymatic oxidative decarboxylation of linear short‐chain fatty acids (C4:0–C9:0) employing the P450 monooxygenase OleT, O₂ as the oxidant, and NAD(P)H as the electron donor gave the corresponding terminal C₃ to C₈ alkenes with product titers of up to 0.93 g L^?1 and TTNs of >2000. Key to this process was the construction of an efficient electron‐transfer chain employing putidaredoxin CamAB in combination with NAD(P)H recycling at the expense of glucose, formate, or phosphite. This system allows for the biocatalytic production of industrially important 1‐alkenes, such as propene and 1‐octene, from renewable resources for the first time.     

Using Small Molecules to Enhance P450 OleT Enzyme Activity in Situ

Cytochrome P450 OleT is a fatty acid decarboxylase that catalyzes the production of olefins with biofuel and synthetic applications. However, the relatively sluggish catalytic efficiency of the enzyme limits its applications. Here, we report the application of a novel class of benzene containing small molecules to improve the OleT activity. The UV-Vis spectroscopy study and molecular docking results confirmed the high proximity of the small molecules to the heme group of OleT. Up to 6-fold increase of product yield has been achieved in the small molecule-modulated enzymatic reactions. Our work thus sheds the light to the application of small molecules to increase the OleT catalytic efficiency, which could be potentially used for future olefin productions.     

Pac13 is a Small,Monomeric Dehydratase that Mediates the Formation of the 3′‐Deoxy Nucleoside of Pacidamycins

The uridyl peptide antibiotics (UPAs), of which pacidamycin is a member, have a clinically unexploited mode of action and an unusual assembly. Perhaps the most striking feature of these molecules is the biosynthetically unique 3′‐deoxyuridine that they share. This moiety is generated by an unusual, small and monomeric dehydratase, Pac13, which catalyses the dehydration of uridine‐5′‐aldehyde. Here we report the structural characterisation of Pac13 with a series of ligands, and gain insight into the enzyme's mechanism demonstrating that H42 is critical to the enzyme's activity and that the reaction is likely to proceed via an E1cB mechanism. The resemblance of the 3′‐deoxy pacidamycin moiety with the synthetic anti‐retrovirals, presents a potential opportunity for the utilisation of Pac13 in the biocatalytic generation of antiviral compounds.     

Mechanism of 3-Methylglutaconyl CoA Decarboxylase AibA/AibB: Pericyclic Reaction versus Direct Decarboxylation

The enzyme 3-methylglutaconyl coenzyme A (CoA) decarboxylase (called AibA/AibB) catalyzes the decarboxylation of 3-methylglutaconyl CoA to generate 3,3-dimethylacrylyl-CoA, representing an important step in the biosynthesis of isovaleryl-coenzyme A in Myxococcus xanthus when the regular pathway is blocked. A novel mechanism involving a pericyclic transition state has previously been proposed for this enzyme, making AibA/AibB unique among decarboxylases. Herein, density functional calculations are used to examine the energetic feasibility of this mechanism. It is shown that the intramolecular pericyclic reaction is associated with a very high energy barrier that is similar to the barrier of the same reaction in the absence of the enzyme. Instead, the calculations show that a direct decarboxylation mechanism has feasible energy barriers that are in line with the experimental observations.     

Pac13 is a Small,Monomeric Dehydratase that Mediates the Formation of the 3′‐Deoxy Nucleoside of Pacidamycins

The uridyl peptide antibiotics (UPAs), of which pacidamycin is a member, have a clinically unexploited mode of action and an unusual assembly. Perhaps the most striking feature of these molecules is the biosynthetically unique 3′‐deoxyuridine that they share. This moiety is generated by an unusual, small and monomeric dehydratase, Pac13, which catalyses the dehydration of uridine‐5′‐aldehyde. Here we report the structural characterisation of Pac13 with a series of ligands, and gain insight into the enzyme's mechanism demonstrating that H42 is critical to the enzyme's activity and that the reaction is likely to proceed via an E1cB mechanism. The resemblance of the 3′‐deoxy pacidamycin moiety with the synthetic anti‐retrovirals, presents a potential opportunity for the utilisation of Pac13 in the biocatalytic generation of antiviral compounds.     

The Biosynthesis and Catabolism of the Maleic Anhydride Moiety of Stipitatonic Acid

A series of directed knockout experiments, combined with an in vitro assay of pathway components, has elucidated for the first time the chemical steps involved in the biosynthesis of the tropolone class of fungal maleic anhydrides. The pathway involves the stepwise oxidation of aldehyde and methyl carbon atoms to form a 1,2‐dicarboxylate. A hydrolase‐catalyzed interconversion of this and the corresponding maleic anhydride, followed by decarboxylation of the diacid leads to the pathway’s final product of stipitatic acid.     

Evolution in microfluidic droplet

High-throughput screening (HTS) of enzymatic activity is important for directed evolution-based enzyme engineering. However, substrate and product diffusion can severely compromise these HTS assays. In this issue of Chemistry & Biology, Kintses and coworkers describe a microfluidic platform for the directed evolution of enzymes in droplets that allows for the screening of 10(7) mutants per round of evolution.     

What can electrochemistry tell us about individual enzymes?

We provide here a critical analysis of electrochemistry's potential and limitations in investigating single-enzyme catalysis, highlighting papers of interest from the past 2–3 years with an emphasis on nano-impact electrochemistry (NIE) and electrochemical scanning tunneling microscopy. NIE can report single-enzyme activity; however, its future broad applicability for studying freely diffusing individual enzymes is questionable. Electrochemical scanning tunneling microscopy, an alternative to NIE, measures single enzyme's electronic conductivity when suspended between two electrodes. Recent discoveries indicate that enzyme conductance depends directly on biophysical parameters such as substrate binding, oxidation state of the catalytic center, and structural fluctuations. We conclude with a short perspective on additional electrochemical routes and combinations of existing techniques that may be useful for studying single-enzyme characteristics.     

酶立体选择性的定向进化及其高通量筛选方法

定向进化技术已成为开发新型生物催化剂的有力工具,特别是在对酶结构或催化机理信息缺乏的情况下。酶的立体选择性是个比较难处理的参数,其在定向进化过程中的技术瓶颈是建立快速有效的高通量筛选方法。本文概述了在酶立体选择性的定向进化方面所取得的进展,着重论述了酶立体选择性的高通量筛选方法。     

Directed evolution of oxygenases: screening systems, success stories and challenges

The field of directed evolution of oxygenases (mono-, di- and epoxygenases) is rapidly advancing as an increasing number of success stories indicate. A significant number of screening systems have been developed to specifically improve oxygenase properties. Oxygenases will become very valuable biocatalysts for synthetic applications in industry when stability, cofactor and activity properties match industrial demands. This review summarizes screening systems and principles of screening systems that have been used for directed evolution of oxygenases. Sections on mutagenic conditions, mutant library size and property improvements provide a comprehensive picture on performance and limitations of current directed evolution methodologies for oxygenases. A discussion of challenges in the directed evolution of oxygenases for industrial exploitation concludes this review.     

Total Synthesis of Dansylated Park's Nucleotide for High‐Throughput MraY Assays

The membrane protein translocase I (MraY) is a key enzyme in bacterial peptidoglycan biosynthesis. It is therefore frequently discussed as a target for the development of novel antibiotics. The screening of compound libraries for the identification of MraY inhibitors is enabled by an established fluorescence‐based MraY assay. However, this assay requires a dansylated derivative of the bacterial biosynthetic intermediate Park's nucleotide as the MraY substrate. Isolation of Park's nucleotide from bacteria and subsequent dansylation only furnishes limited amounts of this substrate, thus hampering the high‐throughput screening for MraY inhibitors. Accordingly, the efficient provision of dansylated Park's nucleotide is a major bottleneck in the exploration of this promising drug target. In this work, we present the first total synthesis of dansylated Park's nucleotide, affording an unprecedented amount of the target compound for high‐throughput MraY assays.     

Towards the Evolution of Artificial Metalloenzymes—A Protein Engineer's Perspective

Incorporating artificial metal‐cofactors into protein scaffolds results in a new class of catalysts, termed biohybrid catalysts or artificial metalloenzymes. Biohybrid catalysts can be modified chemically at the first coordination sphere of the metal complex, as well as at the second coordination sphere provided by the protein scaffold. Protein‐scaffold reengineering by directed evolution exploits the full power of nature's diversity, but requires validated screening and sophisticated metal cofactor conjugation to evolve biohybrid catalysts. In this Minireview, we summarize the recent efforts in this field to establish high‐throughput screening methods for biohybrid catalysts and we show how non‐chiral catalysts catalyze reactions enantioselectively by highlighting the first successes in this emerging field. Furthermore, we shed light on the potential of this field and challenges that need to be overcome to advance from biohybrid catalysts to true artificial metalloenzymes.