Search

Many natural metalloenzymes assemble from proteins and biosynthesised complexes, generating potent catalysts by changing metal coordination. Here we adopt the same strategy to generate artificial metalloenzymes (ArMs) using ligand exchange to unmask catalytic activity. By systematically testing RuII(η6‐arene)(bipyridine) complexes designed to facilitate the displacement of functionalised bipyridines, we develop a fast and robust procedure for generating new enzymes via ligand exchange in a protein that has not evolved to bind such a complex. The resulting metal cofactors form peptidic coordination bonds but also retain a non‐biological ligand. Tandem mass spectrometry and 19F NMR spectroscopy were used to characterise the organometallic cofactors and identify the protein‐derived ligands. By introduction of ruthenium cofactors into a 4‐helical bundle, transfer hydrogenation catalysts were generated that displayed a 35‐fold rate increase when compared to the respective small molecule reaction in solution., A ruthenium organometallic complex is transformed into an effective transfer hydrogenation catalyst upon exchanging ligands with a naïve protein. The direct coordination of protein sidechains to the metal is an underutilised feature in artificial metalloenzymes.

Given the fact that the theoretically possible number of racemates is larger than that of symmetric prochiral or meso compounds,1 the development of deracemization methods, which yield a single stereoisomer from a racemate is an important topic.1–3 Enantioconvergent processes are based on the transformation of a pair of enantiomers through opposite stereochemical pathways affecting retention and inversion of configuration. Depending on the stereochemical course of enzymatic and chemical reactions, three types of deracemization protocols were recently classified by Feringa et al.4 Two chemoenzymatic methods start with a biocatalytic kinetic resolution step, which yields a hetero- or homochiral 1:1 mixture of the formed product and nonconverted substrate enantiomer. The latter is subjected to a second (non-enzymatic) transformation with retention or inversion of configuration to yield a single stereoisomeric product. Although several one-pot, two-step protocols have been successfully demonstrated,5, 10c,d they typically rely on activated species, such as sulfonates,5a–d nitrate esters,5b or Mitsunobu intermediates,5e and negatively affect the overall atom economy of the process. The most elegant method relies on one (or two) enzyme(s), which mediate the transformation of both enantiomers through stereocomplementary pathways by retention and inversion. Since the requirements of such double selectivities are very difficult to meet, successful examples are rare: This approach has been applied to the hydrolysis of epoxides using two epoxide hydrolases showing opposite enantiopreference6 or a single enzyme that catalyzes the enantioconvergent hydrolysis of enantiomers with opposite regioselectivity.7 For enzymes, the ability to act by retention or inversion is a rare feature, which has been found among epoxide hydrolases,8 dehalogenases,4, 9 and sulfatases.10 The latter catalyze the hydrolytic cleavage of (alkyl) sulfate esters by breakage of the S–O or the C–O bond leading to retention or inversion at the chiral carbon atom,10b and thus makes them prime candidates for enantioconvergent processes. So far, only a single inverting sec-alkylsulfatase (PISA1) was generated recombinantly and characterized biochemically,11 thus allowing preparative-scale applications.10c In combination with acid-catalyzed hydrolysis of the nonreacted substrate enantiomer under retention of configuration12 a chemoenzymatic two-step deracemization protocol for sec-alcohols was recently developed.10c,d However, the method suffers from serious limitations because it requires undesirably large volumes organic solvents and several molar equivalents of a strong acid (typically 2–7 equiv of p-TosOH), which pose the risk of racemization or decomposition to the functionalized substrates, especially when elevated temperatures are required for acidic hydrolysis. Moreover, it is not applicable to retaining sulfatases, because no chemical method for sulfate ester hydrolysis with inversion exists.10c So far, retaining-sulfatase activity was reported in whole cells of Rhodopirellula baltica DSM 10527,13 but the corresponding enzymes could not be identified, thus impeding the use of recombinant technology to make the enzyme available for biocatalysis. Furthermore, the retaining sulfatase of Rh. baltica would not be suitable for an enantioconvergent process with PISA1, because both proteins exhibit the same enantiopreference. During our search for a retaining sec-alkylsulfatase with an enantiopreference opposite to that of PISA1, we discovered that the arylsulfatase from Pseudomonas aeruginosa (PAS) exhibited activity on sec-alkylsulfates. PAS, which has been characterized on a molecular level,14 showed promiscuous activity on various arylic phosphates and phosphonates.15 On its standard model substrate (4-nitrophenyl sulfate), PAS exhibited a rate acceleration of kcat/kuncat 2.3×1010,16 and for a less reactive substrate the highest rate enhancement (kcat/kuncat=2×1026) of any catalytic reaction known so far has been measured.17 The stereochemical features of PAS were investigated using a series of sec-alkylsulfate esters (rac-1 a–7 a; Table ​Table1).1). The substrates 1 a–3 a bearing an acetylenic moiety on the long chain adjacent to the stereocenter were resolved with good to excellent enantioselectivities (E 59 to >200). Undesired non-enzymatic background hydrolysis of 1 a could be suppressed by addition of 20 % (v/v) of DMSO as a cosolvent.18 In contrast, the selectivities were largely lost when the acetylenic moiety was moved to the short chain (substrates 4 a–6 a). The alkyl aryl derivative 7 a gave again an excellent E value of greater than 200. All substrates converted with high enantioselectivities (1 a–3 a, 7 a) were hydrolyzed with complete retention of configuration, thus yielding S-configured alcohols and unreacted R-configured sulfate esters. To prove the stereochemical course of sulfate ester hydrolysis by PAS, enzymatic cleavage of rac-1-octyn-3-yl sulfate (6 a) was performed in an 18O-labeled buffer (label >98 %). GC/MS analysis of the alcohol 6 b formed revealed that (in contrast to inverting sec-alkylsulfatases10c) no incorporation of 18O occurred, and is consistent with the attack of the enzyme’s formylglycine nucleophile on sulfur. Hydrolysis of enantiopure (S)-6 a by PAS yielded alcohol (S)-6 b in greater than 99 % ee, thus proving that hydrolysis proceeded under strict retention of configuration. Table 1 Kinetic resolution of sulfate esters rac-1 a–7 a with retention and inversion using PAS and PISA1 Since the stereochemical features of PAS—R enantiopreference with retention—would ideally complement the S preference with inversion10c,d of PISA1,10e we optimized the enzymatic hydrolysis of the substrates 1 a–7 a with the latter enzyme (Table ​(Table1).1). Most of the substrates (1 a, 4 a–7 a) showed perfect E values of greater than 200, with only 2 a and 3 a giving insufficient selectivities. The data thus obtained enabled us to develop three types of enantioconvergent processes (Scheme 1, Table ​Table22): Table 2 Deracemization of the sulfate esters rac-1 a–7 a using retaining PAS and inverting PISA1. Scheme 1 One-pot, two-enzyme deracemization process using retaining PAS and inverting PISA1. Type A. The substrates rac-2 a and rac-3 a, where PAS was highly enantioselective, could be deracemized by a one-pot, two-step sequence using retaining PAS first, followed by non-enantioselective inverting hydrolysis with PISA1 to yield (S)-2 b and (S)-3 b in 91 and 97 % ee, respectively. Type B. For rac-4 a–6 a, where PISA1 was highly enantioselective, but PAS was not, the opposite order of events—PISA1 first, PAS second—was successful and yielded the corresponding R-configured alcohols 4 b–6 b in 94 to greater than 99 % ee Type C. The ideal single-step process using both enzymes simultaneously was designed for the substrates rac-1 a and rac-7 a. To maximize the ee value of 1 b, DMSO was used as cosolvent to suppress background hydrolysis which increased the ee value of (S)-1 b from 93 to 98 % ee. To demonstrate the applicability of this method on a preparative scale, the deracemization of rac-6 a was scaled-up (1 g, 4.4 mmol), and gave (R)-6 b as the sole product in 82 % yield upon isolation with 98 % ee. The choice of which process (Type A–C) is most suitable for the deracemization of a given substrate depends on the availability of an enantioselective sec-alkylsulfatase acting with retention or inversion of configuration. Processes of Types A and B are feasible with a single enantioselective enzyme, whereas Type C requires two enantioselective sulfatases with matching opposite enantiopreference. It should be kept in mind that Types A and B constitute kinetic resolutions,19 whereas Type C represents a parallel kinetic resolution.20 Overall, the purely enzymatic protocol excels by its significantly broader applicability compared to the chemoenzymatic procedure10c,d for the following reasons: 1) it eliminates the harsh reaction conditions required for acid-catalyzed hydrolysis, which are incompatible with sensitive functional groups and 2) it is also applicable to retaining sulfatases (such as PAS). In summary, the one-pot deracemization of sec-alcohols bearing various functional groups was achieved by enantioconvergent hydrolysis of the corresponding sulfate esters using the retaining aryl sulfatase PAS and the inverting alkyl sulfatase PISA1, which possess the required opposite enantiopreference.

Efficient delivery of small interfering RNA (siRNA) into cells is the basis of target-gene-specific silencing and, ultimately, gene therapy. However, current transfection reagents are relatively inefficient, and very few studies provide the sort of systematic understanding based on structure-activity relationships that would provide rationales for their improvement. This work established peptide dendrimers (administered with cationic lipids) as siRNA transfection reagents and recorded structure-activity relationships that highlighted the importance of positive charge distribution in the two outer layers and a hydrophobic core as key features for efficient performance. These dendrimer-based transfection reagents work as well as highly optimised commercial reagents, yet show less toxicity and fewer off-target effects. Additionally, the degrees of freedom in the synthetic procedure will allow the placing of decisive recognition features to enhance and fine-tune transfection and cell specificity in the future.

Best of both worlds: The interplay of active site reactivity and the dynamic character of proteins allows enzymes to be promiscuous and--sometimes--remarkably efficient at the same time. This review analyses the roles structural flexibility and chemical reactivity play in the catalytic mechanism of selected enzymes.

Developmental cell biology requires technologies in which the fate of single cells is followed over extended time periods, to monitor and understand the processes of self-renewal, differentiation, and reprogramming. A workflow is presented, in which single cells are encapsulated into droplets (Ø: 80 µm, volume: ≈270 pL) and the droplet compartment is later converted to a hydrogel bead. After on-chip de-emulsification by electrocoalescence, these 3D scaffolds are subsequently arrayed on a chip for long-term perfusion culture to facilitate continuous cell imaging over 68 h. Here, the response of murine embryonic stem cells to different growth media, 2i and N2B27, is studied, showing that the exit from pluripotency can be monitored by fluorescence time-lapse microscopy, by immunostaining and by reverse-transcription and quantitative PCR (RT-qPCR). The defined 3D environment emulates the natural context of cell growth (e.g., in tissue) and enables the study of cell development in various matrices. The large scale of cell cultivation (in 2000 beads in parallel) may reveal infrequent events that remain undetected in lower throughput or ensemble studies. This platform will help to gain qualitative and quantitative mechanistic insight into the role of external factors on cell behavior.

Mikrotropfchen in Mikrofluidiksystemen bieten zahlreiche Moglichkeiten fur die chemische und biologische Forschung. Sie ermoglichen die isolierte Betrachtung von Spezies oder Reaktionen, sie sind monodispers und daher fur quantitative Studien geeignet, sie konnen fur Studien in extrem kleinen Volumina sowie an einzelnen Zellen oder einzelnen Molekulen eingesetzt werden, und sie sind fur Hochdurchsatzexperimente geeignet. Dieser Aufsatz analysiert die Bedeutung dieser Eigenschaften im Hinblick auf neue biologische und chemische Experimente, wobei jungste Fortschritte bei der Entwicklung von Apparaturen vorgestellt, aber auch verbleibende technologische Herausforderungen angesprochen werden. Anhand von Beispielen wird gezeigt, welche Vorteile die Kompartimentierung, die Monodispersitat der Tropfchen, die Betrachtung einzelner Molekule und der hohe Durchsatz in Experimenten gebracht haben, die ohne den Einsatz von Mikrofluidiksystemen kaum moglich gewesen waren.

The systematic exploration of the modification of polyethylene imine with guanidinium and octyl groups has led to the identification of a catalyst, CD6, which accelerates the phosphate transfer reaction of HPNP (2-hydroxypropyl-4-nitrophenyl phosphate) in the presence of divalent metals such as Zn(2+), Co(2+), Mg(2+) or Ni(2+). CD6 exhibits saturation kinetics that are described by Michaelis-Menten parameters K(m) ranging from 2.5-8 mM and k(cat) ranging from 0.0014-0.09 s(-1). For Zn(II)-CD6 this corresponds to an overall acceleration k(cat)/k(uncat) of 3.8x10(5) and a catalytic proficiency (k(cat)/K(m))/k(uncat) of 1.5x10(8). Catalysis by Zn(II)-CD6 is specifically inhibited by inorganic phosphate, allowing turnover regulation by product inhibition. This effect stands in contrast to Zn(II)-catalysed transesterification of HPNP in water or by the synzymes Co(II)-CD6 and Ni(II)-CD6, with which no such interference by product is observed. These characteristics render synzyme Zn(II)-CD6 an efficient enzyme model that reflects enzyme-like properties in a wide range of features.

The catalytic potential of tyrocidine synthetase 1 (TycA) was probed by the kinetic characterization of its adenylation activity. We observed reactions with 30 substrates, thus suggesting some substrate promiscuity. However, although the TycA adenylation (A) domain was able to accommodate alternative substrates, their k(cat)/K(M) values ranged over six orders of magnitude. A comparison of the activities allowed the systematic mapping of the substrate specificity determinants of the TycA A-domain. Hydrophobicity plays a major role in the recognition of substrate analogues but can be combined with shape complementarity, conferring higher activity, and/or steric exclusion, leading to substantial discrimination against larger substrates. A comparison of the k(cat)/K(M) values of the TycA A-domain and phenylalanyl-tRNA synthetase showed that the level of discrimination was comparable in the two enzymes for the adenylation reaction and suggested that TycA was also subjected to high selective pressure. The specificity patterns observed and the quantification of alternative activities provide a basis for exploring possible paths for the future directed evolution of A-domain specificity.

Histone acetylation plays an integral role in the epigenetic regulation of gene expression. Transcriptional activity reflects the recruitment of opposing classes of enzymes to promoter elements; histone acetyltransferases (EC 2.3.1.48) that deposit acetyl marks at a subset of histone residues and histone deacetylases that remove them. Many histone acetyltransferases are difficult to study in solution because of their limited stability once purified. We have developed a directed evolution protocol that allows the screening of hundreds of histone acetyltransferase mutants for histone acetylating activity, and used this to enhance the thermostability of the human P/CAF histone acetyltransferase. Two rounds of directed evolution significantly stabilized the enzyme without lowering the catalytic efficiency and substrate specificity of the enzyme. Twenty-four variants with higher thermostability were identified. Detailed analysis revealed twelve single amino acid mutants that were found to possess a higher thermostability. The residues affected are scattered over the entire protein structure, and are different from mutations predicted by sequence alignment approaches, suggesting that sequence comparison and directed evolution methods are complementary strategies in engineering increased protein thermostability. The stabilizing mutations are predominately located at surface of the enzyme, suggesting that the protein's surface is important for stability. The directed evolution approach described in the present study is easily adapted to other histone modifying enzymes, requiring only appropriate peptide substrates and antibodies, which are available from commercial suppliers.

Reference LIP-ARTICLE-2008-008doi:10.1002/cbic.200700459View record in Web of Science Record created on 2008-06-09, modified on 2017-05-12

Best of both worlds: The interplay of active site reactivity and the dynamic character of proteins allows enzymes to be promiscuous and--sometimes--remarkably efficient at the same time. This review analyses the roles structural flexibility and chemical reactivity play in the catalytic mechanism of selected enzymes.

Microdroplets in microfluidics offer a great number of opportunities in chemical and biological research. They provide a compartment in which species or reactions can be isolated, they are monodisperse and therefore suitable for quantitative studies, they offer the possibility to work with extremely small volumes, single cells, or single molecules, and are suitable for high-throughput experiments. The aim of this Review is to show the importance of these features in enabling new experiments in biology and chemistry. The recent advances in device fabrication are highlighted as are the remaining technological challenges. Examples are presented to show how compartmentalization, monodispersity, single-molecule sensitivity, and high throughput have been exploited in experiments that would have been extremely difficult outside the microfluidics platform.

MIMICKING the efficiency of enzyme catalysis is a daunting challenge. An enzyme selectively binds and stabilizes the transition state(s) for a particular reaction1,2. Artificial host systems can bind ground states just as efficiently3, and rate enhancements comparable to those in enzymatic reactions can be achieved by bringing catalytic and substrate groups together in intramolecular reactions. But the combination of selective binding and efficient catalysis remains elusive. The best enzyme mimics currently known are catalytic antibodies5,6. They bind transition-state analogues with high affinity, but their catalytic efficiency generally falls far short of that of enzymes4,8. Thorn et al.9 recently described an antibody that catalyses the eliminative ring-opening of a benzisoxazole "exceptionally efficiently" using car-boxylate as the general base, raising the intriguing possibility that this high efficiency derives from precise positioning of catalytic and substrate groups10. Here we show that familiar 'off-the-shelf proteins—serum albumins—catalyse the same reaction at similar rates, using a lysine side-chain amino group as the catalytic general base. Comparisons suggest that formal general base catalysis is of only modest efficiency in both systems, and that the antibody catalysis is boosted by a non-specific medium effect.