Biochemical Characterization and Mechanistic Analysis of the Levoglucosan Kinase from Lipomyces starkeyi

Levoglucosan kinase (LGK) catalyzes the simultaneous hydrolysis and phosphorylation of levoglucosan (1,6‐anhydro‐β‐d ‐glucopyranose) in the presence of Mg²⁺–ATP. For the Lipomyces starkeyi LGK, we show here with real‐time in situ NMR spectroscopy at 10 °C and pH 7.0 that the enzymatic reaction proceeds with inversion of anomeric stereochemistry, resulting in the formation of α‐d ‐glucose‐6‐phosphate in a manner reminiscent of an inverting β‐glycoside hydrolase. Kinetic characterization revealed the Mg²⁺ concentration for optimum activity (20–50 mm ), the apparent binding of levoglucosan (K_m=180 mm ) and ATP (K_m=1.0 mm ), as well as the inhibition by ADP (K_i=0.45 mm ) and d ‐glucose‐6‐phosphate (IC₅₀=56 mm ). The enzyme was highly specific for levoglucosan and exhibited weak ATPase activity in the absence of substrate. The equilibrium conversion of levoglucosan and ATP lay far on the product side, and no enzymatic back reaction from d ‐glucose‐6‐phosphate and ADP was observed under a broad range of conditions. 6‐Phospho‐α‐d ‐glucopyranosyl fluoride and 6‐phospho‐1,5‐anhydro‐2‐deoxy‐d ‐arabino‐hex‐1‐enitol (6‐phospho‐d ‐glucal) were synthesized as probes for the enzymatic mechanism but proved inactive with the enzyme in the presence of ADP. The pyranose ring flip ⁴C₁→¹C₄ required for 1,6‐anhydro‐product synthesis from d ‐glucose‐6‐phosphate probably presents a major thermodynamic restriction to the back reaction of the enzyme.     

Structural basis for the properties of two single-site proline mutants of CYP102A1 (P450BM3)

The crystal structures of the haem domains of Ala330Pro and Ile401Pro, two single‐site proline variants of CYP102A1 (P450_BM3) from Bacillus megaterium, have been solved. In the A330P structure, the active site is constricted by the relocation of the Pro329 side chain into the substrate access channel, providing a basis for the distinctive C? H bond oxidation profiles given by the variant and the enhanced activity with small molecules. I401P, which is exceptionally active towards non‐natural substrates, displays a number of structural similarities to substrate‐bound forms of the wild‐type enzyme, notably an off‐axial water ligand, a drop in the proximal loop, and the positioning of two I‐helix residues, Gly265 and His266, the reorientation of which prevents the formation of several intrahelical hydrogen bonds. Second‐generation I401P variants gave high in vitro oxidation rates with non‐natural substrates as varied as fluorene and propane, towards which the wild‐type enzyme is essentially inactive. The substrate‐free I401P haem domain had a reduction potential slightly more oxidising than the palmitate‐bound wild‐type haem domain, and a first electron transfer rate that was about 10 % faster. The electronic properties of A330P were, by contrast, similar to those of the substrate‐free wild‐type enzyme.     

The 2-oxoglutarate-dependent oxygenase JMJD6 catalyses oxidation of lysine residues to give 5S-hydroxylysine residues

Amino acid analyses reveal that JMJD6-catalysed hydroxylation of RNA-splicing regulatory protein fragments occurs to give hydroxylysine products with 5S stereochemistry. This contrasts with collagen lysyl hydroxylases, which give 5R-hydroxylated products. The work suggests that more than one subfamily of lysyl hydroxylases has evolved and illustrates the importance of stereochemical assignments in proteomic analyses.     

Molecular Aspects of Catalytic Reactivity. Application of EPR Spectroscopy to Stuies of the Mechanism of Heterogeneous Catalytic Reactions

The basic objective of mechanistic studies of real catalytic processes is to dissect the course of the reaction into individual steps; ascertain their sequence; and determine the stoichiometry, structure, and electronic states of active sites and intermediates. The electron paramagnetic resonance (EPR) technique is at present widely used to explore many of these principal aspects of heterogeneous catalysis and surface chemistry. The extreme sensitivity compared to the usual spectroscopic methods is perhaps its most acknowledged advantage and makes EPR best suited to investigate and characterize low-abundance active sites and intermediates appearing during catalytic reaction. Additional information can be drawn from the theoretical analysis of the experimental spin Hamiltonian parameters within the ligand field and from angular overlap or Newman's superposition models as well as by more sophisticated quantum chemical calculations. The purpose of this paper is to show how catalysis benefits from EPR spectroscopy and to identify the issues and areas explored by this method. A comprehensive literature review is not attempted in this article; instead, attention is directed toward application of EPR for elucidation of the molecular reaction mechanism that can provide a scientific background for understanding many fundamental aspects of catalytic activity. The major events of mechanistic studies which involve the identification of active sites, activation of reagents, and determination of the reaction pathways are illustrated by selected examples and discussed. An approach that is complementary to mechanistic catalytic test studies is also presented. It consists of spectroscopic investigations of a set of partial reactions, driven by external creation of the supposed active sites and intermediates, with the aim of reproducing and verifying the feasibility of the postulated catalytic cycle. Moreover, to assure some consistency of the subject, basic characteristics of EPR spectroscopy related to surface studies and chemical theories of reactivity are concisely reviewed.     

Characterization of the Rv3377c Gene Product,a Type‐B Diterpene Cyclase,from the Mycobacterium tuberculosis H37 Genome

The Rv3377c gene from the Mycobacterium tuberculosis H37 genome is specifically limited to those Mycobacterium species that cause tuberculosis. We have demonstrated that the gene product of Rv3377c is a diterpene cyclase that catalyzes the formation of tuberculosinol from geranylgeranyl diphosphate (GGPP). However, the characteristics of this enzyme had not previously been studied in detail with homogeneously purified enzyme. The purified enzyme catalyzed the synthesis of tuberculosinyl diphosphate from GGPP, but it did not bring about the synthesis of tuberculosinol. Optimal conditions for the highest activity were found to be as follows: pH 7.5, 30 °C, Mg^II (0.1 mM ), and Triton X‐100 (0.1 %). Under these conditions, the kinetic values of K_M and k_cat were determined to be 11.7±1.9 μM for GGPP and 12.7±0.7 min^?1, respectively, whereas the specific activity was 186 nmol min^?1 mg^?1. The enzyme activity was inhibited at substrate concentrations higher than 50 μM . The catalytic activity was strongly inhibited by 15‐aza‐dihydrogeranylgeraniol and 5‐isopropyl‐N,N,N,2‐tetramethyl‐4‐(piperidine‐1‐carbonyloxy)benzenaminium chloride (Amo‐1618). The DXDTT_293–297 motif, corresponding to the DXDDTA motif conserved among terpene cyclases, was mutated in order to investigate its function. The middle D295 was found to be the most crucial entity for the catalysis. D293 and two threonine residues function synergistically to enhance the acidity of D295, possibly through hydrogen‐bonding networks. The Rv3377c enzyme could also react with (14R/S)‐14,15‐oxidoGGPP to generate 3α‐ and 3β‐hydroxytuberculosinyl diphosphate. Conformational analyses were carried out with deuterium‐labeled GGPP and oxidoGGPP. We found that GGPP and (14R)‐oxidoGGPP adopted a chair/chair conformation, but (14S)‐oxidoGGPP adopted a boat/chair conformation. Interestingly, the conformations of oxidoGGPP for the A‐ring formation are the opposite of those of oxidosqualene when it is used as a substrate by squalene cyclases for the biosynthesis of hopene and tetrahymanol. (3R)‐Oxidosqualene is folded in a boat conformation, whereas (3S)‐2,3‐oxidosqualene folds into a chair conformation, for the formation of the A‐rings of the hopene and tetrahymanol skeletons, respectively.     

Intramolecular electron transfer in the dihaem cytochrome c peroxidase of Pseudomonas aeruginosa

Mutant forms of the enzyme cytochrome c peroxidase from Pseudomonas aeruginosa, in which the peroxidatic haem ligand (H71) and putative haem-bridging amino acid (W94) have been mutated, were produced in an E. coli expression system as a means of investigating possible mechanisms of intramolecular electron transfer within the enzyme. EPR spectroscopy indicated the presence of a high-spin, presumably five-coordinate, peroxidatic haem site in the H71G and H71G/W94A mutants, whilst the W94A mutant apparently retained the normal six-coordinate haem structures. In turnover experiments, these mutants show 55, 4, and <1% activity, respectively, as compared to the wild-type enzyme. The W94A mutant shows essentially no activity in turnover experiments. Circular dichroism spectroscopy indicates no measurable difference in the secondary structure of the H71G mutant from that of the native enzyme, whilst some small differences are observed for the double mutant. Treatment of the oxidised mutant proteins with hydrogen peroxide, in the absence of preactivation or exogenous reductants, yields products that suggest the formation of a tryptophan radical species in the case of the H71 mutant and the production of a porphyrin radical in the case of the double mutant. These results are discussed in terms of the intramolecular electron transfer in this enzyme.     

Molecular Aspects of Catalytic Reactivity. Application of EPR Spectroscopy to Stuies of the Mechanism of Heterogeneous Catalytic Reactions

The basic objective of mechanistic studies of real catalytic processes is to dissect the course of the reaction into individual steps; ascertain their sequence; and determine the stoichiometry, structure, and electronic states of active sites and intermediates. The electron paramagnetic resonance (EPR) technique is at present widely used to explore many of these principal aspects of heterogeneous catalysis and surface chemistry. The extreme sensitivity compared to the usual spectroscopic methods is perhaps its most acknowledged advantage and makes EPR best suited to investigate and characterize low-abundance active sites and intermediates appearing during catalytic reaction. Additional information can be drawn from the theoretical analysis of the experimental spin Hamiltonian parameters within the ligand field and from angular overlap or Newman's superposition models as well as by more sophisticated quantum chemical calculations. The purpose of this paper is to show how catalysis benefits from EPR spectroscopy and to identify the issues and areas explored by this method. A comprehensive literature review is not attempted in this article; instead, attention is directed toward application of EPR for elucidation of the molecular reaction mechanism that can provide a scientific background for understanding many fundamental aspects of catalytic activity. The major events of mechanistic studies which involve the identification of active sites, activation of reagents, and determination of the reaction pathways are illustrated by selected examples and discussed. An approach that is complementary to mechanistic catalytic test studies is also presented. It consists of spectroscopic investigations of a set of partial reactions, driven by external creation of the supposed active sites and intermediates, with the aim of reproducing and verifying the feasibility of the postulated catalytic cycle. Moreover, to assure some consistency of the subject, basic characteristics of EPR spectroscopy related to surface studies and chemical theories of reactivity are concisely reviewed.     

Substrate‐Determined Diastereoselectivity in an Enzymatic Carboligation

Thiamine diphosphate‐dependent enzymes catalyze the formation of C?C bonds, thereby generating chiral secondary or tertiary alcohols. By the use of vibrational circular dichroism (VCD) spectroscopy we studied the stereoselectivity of carboligations catalyzed by YerE, a carbohydrate‐modifying enzyme from Yersinia pseudotuberculosis. Conversion of the non‐physiological substrate (R)‐3‐methylcyclohexanone led to an R,R‐configured tertiary alcohol (diastereomeric ratio (dr) >99:1), whereas the corresponding reaction with the S enantiomer gave the S,S‐configured product (dr>99:1). This suggests that YerE‐catalyzed carboligations can undergo either an R‐ or an S‐specific pathway. We show that, in this case, the high stereoselectivity of the YerE‐catalyzed reaction depends on the substrate's preference to acquire a low‐energy conformation.     

Cooperative Asymmetric Catalysis Using Thioamides toward Truly Practical Organic Syntheses

Asymmetric catalysis has established its unwavering position in organic chemistry as a particularly efficient strategy for the production of enantiomerically enriched compounds. For a truly practical synthetic protocol, however, asymmetric catalysis must be endowed with high atom economy and sustainability. Cooperative activation is a powerful strategy to drive efficient asymmetric catalysis without the use of stoichiometric activating reagents, leading to asymmetric catalysis with minimum waste production. This review summarizes the utility of thioamides as an emerging functional group class in cooperative asymmetric catalysis. Soft Lewis acid/hard Brønsted base cooperative catalysts, in which thioamides serve as either electrophiles or pronucleophiles, promote asymmetric C C bond-forming reactions with perfect atom economy.     

Driving Force Analysis of Proton Tunnelling Across a Reactivity Series for an Enzyme‐Substrate Complex

Quantitative structure‐activity relationships are widely used to probe C? H bond breakage by quinoprotein enzymes.^[1–4] However, we showed recently that p‐substituted benzylamines are poor reactivity probes for the quinoprotein aromatic amine dehydrogenase (AADH) because of a requirement for structural change in the enzyme‐substrate complex prior to C? H bond breakage.^[5] This rearrangement is partially rate limiting, which leads to deflated kinetic isotope effects for p‐substituted benzylamines. Here we report reactivity (driving force) studies of AADH with p‐substituted phenylethylamines for which the kinetic isotope effect (～16) accompanying C? H/C? ²H bond breakage is elevated above the semi‐classical limit. We show bond breakage occurs by quantum tunnelling and that within the context of the environmentally coupled framework for H‐tunnelling the presence of the p‐substituent places greater demand on the apparent need for fast promoting motions. The crystal structure of AADH soaked with phenylethylamine or methoxyphenylethylamine indicates that the structural change identified with p‐substituted benzylamines should not limit the reaction with p‐substituted phenylethylamines. This is consistent with the elevated kinetic isotope effects measured with p‐substituted phenylethylamines. We find a good correlation in the rate constant for proton transfer with bond dissociation energy for the reactive C? H bond, consistent with a rate that is limited by a Marcus‐like tunnelling mechanism. As the driving force becomes larger, the rate of proton transfer increases while the Marcus activation energy becomes smaller. This is the first experimental report of the driving force perturbation of H‐tunnelling in enzymes using a series of related substrates. Our study provides further support for proton tunnelling in AADH.     

A Practical NMR‐Based High‐Throughput Assay for Screening Enantioselective Catalysts and Biocatalysts

Two NMR‐based approaches for high‐throughput screening of enantioselective catalysts and biocatalysts are described. One version makes use of pseudo‐enantiomers or pseudo‐meso‐compounds based on ¹³C‐labeling. A throughput of at least 1400 ee determinations per day is possible by using an appropriate flow‐through cell and an autosampler. The other approach is based on traditional diastereomer formation using a chiral reagent or complexing agent. The ee values are accurate to within ±2% and ±5% of the true values.     

Concerted Electron/Proton Transfer Mechanism in the Oxidation of Phenols by Laccase

This study aimed to assess structural requirements in the enzyme/substrate interactions that are responsible for tuning the enzymatic reactivity. To better assess the role of the aspartic residue in the substrate‐binding pocket of basidiomycete‐type laccases, we compared the catalytic efficiency of wild‐type enzymes to that of a mutant in which carboxylic acid residue Asp206 was changed to alanine. Oxidation efficiency towards phenolic substrates by laccases of Trametes villosa, Trametes versicolor and a T. versicolor D206A mutant was studied at two pH values. By the Hammett approach and Marcus analysis, we obtained unambiguous evidence that the oxidation takes place by a concerted electron/proton transfer (EPT) mechanism, and that at pH 5 (optimum pH for enzyme activity) the phenolic proton is transferred to Asp206 during the concerted electron/proton transfer process.