Curcumin degradation in a soil microorganism: Screening and characterization of a ß-diketone hydrolase.

Curcumin is a plant-derived secondary metabolite exhibiting antitumor, neuroprotective, antidiabetic activities, and so on. We previously isolated Escherichia coli as an enterobacterium exhibiting curcumin-converting activity from human feces, and discovered an enzyme showing this activity (CurA) and named it NADPH-dependent curcumin/dihydrocurcumin reductase. From soil, here, we isolated a curcumin-degrading microorganism (No. 34) using the screening medium containing curcumin as the sole carbon source and identified as Rhodococcus sp. A curcumin-degrading enzyme designated as CurH was purified from this strain and characterized, and compared with CurA. CurH catalyzed hydrolytic cleavage of a carbon-carbon bond in the ß-diketone moiety of curcumin and its analogs, yielding two products bearing a methyl ketone terminus and a carboxylic acid terminus, respectively. These findings demonstrated that a curcumin degradation reaction catalyzed by CurH in the soil environment was completely different from the one catalyzed by CurA in the human microbiome. Of all the curcumin analogs tested, suitable substrates for the enzyme were curcuminoids (i.e., curcumin and bisdemethoxycurcumin) and tetrahydrocurcuminoids. Thus, we named this enzyme curcuminoid hydrolase. The deduced amino acid sequence of curH exhibited similarity to those of members of acetyl-CoA C-acetyltransferase family. Considering results of oxygen isotope analyses and a series of site-directed mutagenesis experiments on our enzyme, we propose a possible catalytic mechanism of CurH, which is unique and distinct from those of enzymes degrading ß-diketone moieties such as ß-diketone hydrolases known so far.

Synthesis of Biosynthetic Intermediates of Vibrioferrin and Enzyme Reactions Using Them as Substrates.

Biosynthetic intermediates of siderophore vibrioferrin (VF), O-citryl-L-serine, 2-aminoethyl citrate, and alanine-2-amidoethyl citrate were respectively synthesized as a mixture of stereoisomers. These compounds were used as substrates for enzyme reactions using recombinant PvsA, PvsB, and PvsE proteins as corresponding enzyme equivalents. The results of our study show that each enzyme reacts with a respective substrate and produces VF along the proposed biosynthetic pathway. Furthermore, the results of this study will contribute to the understanding of VF biosynthetic enzymes and may help in the development of antimicrobial drugs by inhibiting siderophore biosynthetic enzymes.

The Potential Roles of Transacylation in Intracellular Lipolysis and Related Qssa Approximations.

Fatty acids (FAs) are crucial energy metabolites, signalling molecules, and membrane building blocks for a wide range of organisms. Adipose triglyceride lipase (ATGL) is the first and presumingly most crucial regulator of FA release from triacylglycerols (TGs) stored within cytosolic lipid droplets. However, besides the function of releasing FAs by hydrolysing TGs into diacylglycerols (DGs), ATGL also promotes the transacylation reaction of two DG molecules into one TG and one monoacylglycerol molecule. To date, it is unknown whether DG transacylation is a coincidental byproduct of ATGL-mediated lipolysis or whether it is physiologically relevant. Experimental evidence is scarce since both, hydrolysis and transacylation, rely on the same active site of ATGL and always occur in parallel in an ensemble of molecules. This paper illustrates the potential roles of transacylation. It shows that, depending on the kinetic parameters but also on the state of the hydrolytic machinery, transacylation can increase or decrease downstream products up to 80% respectively 30%. We provide an extensive asymptotic analysis including quasi-steady-state approximations (QSSA) with higher order correction terms and provide numerical simulation. We also argue that when assessing the validity of QSSAs one should include parameter sensitivity derivatives. Our results suggest that the transacylation function of ATGL is of biological relevance by providing feedback options and altogether stability to the lipolytic machinery in adipocytes.

Demystifying DPP III Catalyzed Peptide Hydrolysis-Computational Study of the Complete Catalytic Cycle of Human DPP III Catalyzed Tynorphin Hydrolysis.

Dipeptidyl peptides III (DPP III) is a dual-domain zinc exopeptidase that hydrolyzes peptides of varying sequence and size. Despite attempts to elucidate and narrow down the broad substrate-specificity of DPP III, there is no explanation as to why some of them, such as tynorphin (VVYPW), the truncated form of the endogenous heptapeptide spinorphin, are the slow-reacting substrates of DPP III compared to others, such as Leu-enkephalin. Using quantum molecular mechanics calculations followed by various molecular dynamics techniques, we describe for the first time the entire catalytic cycle of human DPP III, providing theoretical insight into the inhibitory mechanism of tynorphin. The chemical step of peptide bond hydrolysis and the substrate binding to the active site of the enzyme and release of the product were described for DPP III in complex with tynorphin and Leu-enkephalin and their products. We found that tynorphin is cleaved by the same reaction mechanism determined for Leu-enkephalin. More importantly, we showed that the product stabilization and regeneration of the enzyme, but not the nucleophilic attack of the catalytic water molecule and inversion at the nitrogen atom of the cleavable peptide bond, correspond to the rate-determining steps of the overall catalytic cycle of the enzyme.

Mechanistic Aspects for the Modulation of Enzyme Reactions on the DNA Scaffold.

Cells have developed intelligent systems to implement the complex and efficient enzyme cascade reactions via the strategies of organelles, bacterial microcompartments and enzyme complexes. The scaffolds such as the membrane or protein in the cell are believed to assist the co-localization of enzymes and enhance the enzymatic reactions. Inspired by nature, enzymes have been located on a wide variety of carriers, among which DNA scaffolds attract great interest for their programmability and addressability. Integrating these properties with the versatile DNA-protein conjugation methods enables the spatial arrangement of enzymes on the DNA scaffold with precise control over the interenzyme distance and enzyme stoichiometry. In this review, we survey the reactions of a single type of enzyme on the DNA scaffold and discuss the proposed mechanisms for the catalytic enhancement of DNA-scaffolded enzymes. We also review the current progress of enzyme cascade reactions on the DNA scaffold and discuss the factors enhancing the enzyme cascade reaction efficiency. This review highlights the mechanistic aspects for the modulation of enzymatic reactions on the DNA scaffold.

AFM-based single-molecule observation of the conformational changes of DNA structures.

Direct visualization of the biomolecules of interest is a straightforward way to elucidate the physical properties of individual molecules and their reaction processes. Atomic force microscopy (AFM) enables direct imaging of biomolecules in suitable solution conditions. As AFM visualizes the molecules at a nanometer-scale spatial resolution, a versatile observation platform is required for precise imaging of the molecules in action. The DNA origami technology allows precise placement of target molecules in a designed nanostructure, enabling their detection at the single-molecule level. We used DNA origami technology for visualizing the detailed movement of target molecules in reactions using high-speed AFM (HS-AFM), which enables the analysis of dynamic movement of biomolecules with a subsecond time resolution. By combining the DNA origami system and HS-AFM, DNA conformational changes, including G-quadruplex formation and disruption and B-Z transition, were visualized. In addition, enzyme-based reactions such as DNA recombination were also visualized at the single-molecule level using this combined observation system. Moreover, the enzyme-based reaction could be directly regulated in the DNA origami frame by imposing structural stress on the substrate DNAs to elucidate the reaction mechanism. These target-orientated observation systems should contribute to a detailed analysis of biomolecular motions in real time at molecular resolution.

A nanoplatform based on metal-organic frameworks and coupled exonuclease reaction for the fluorimetric determination of T4 polynucleotide kinase activity and inhibition.

A nanoplatform based on metal-organic frameworks (MOFs) and lambda exonuclease (λ exo) for the fluorimetric determination of T4 polynucleotide kinase (T4 PNK) activity and inhibition is described. Fe-MIL-88 was selected as the nanomaterial because of its significant preferential binding ability to single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA) and its quenching property. The synthesized Fe-MIL-88 was characterized by transmission electron microscope, scanning electron microscope, and X-ray photoelectron spectroscopy. In the presence of T4 PNK, FAM-labeled dsDNA (FAM-dsDNA) is phosphorylated on its 5'-terminal. λ exo then recognizes and cleaves the phosphorylated strand yielding FAM-labeled ssDNA (FAM-ssDNA). The fluorescence of the produced FAM-ssDNA is quenched due to Fe-MIL-88's absorbing on FAM-ssDNA. On the contrary, in the absence of T4 PNK, the phosphorylation and cleavage processes cannot take place. Therefore, the fluorescence of FAM-dsDNA still remains. The fluorescence intensity is detected at the maximum emission wavelength of 524 nm using the maximum excitation wavelength of 488 nm. The assay of T4 PNK based on the fluorescence quenching of FAM-ssDNA achieves a linear relationship in the range 0.01-5.0 U mL-1 with a detection limit of 0.0089 U mL-1 in buffer. The assay exhibits excellent performance for T4 PNK activity determination in a complex biological matrix. The results also reveal the ability of the assay for T4 PNK inhibitor screening. Graphical abstract Schematic presentation of a nanoplatform based on Fe-MIL-88 and coupled exonuclease reaction for the fluorimetric determination of T4 polynucleotide kinase activity. FAM-ssDNA, FAM-labeled single-stranded DNA; cDNA, complementary DNA; λ exo, lambda exonuclease;T4 PNK, T4 polynucleotide kinase.

Viscous Media Slow Down the Decay of the Key Intermediate in Bacterial Bioluminescent Reaction.

The effects of medium viscosity on the decay rate of the 4a-hydroperoxyflavin intermediate of the bioluminescent reaction was investigated. It was found that at low concentrations of glycerol or sucrose (viscosity 1.1-1.3 cP) the decay rate rises, whereas a further increase in viscosity to 6.2 cP leads to a decrease in the decay rate following a power function with an exponent of 0.82-0.84. Using molecular dynamics methods, it was shown that the presence of glycerol and sucrose molecules causes a change in the mobility of the amino acid residues in the active center of luciferase, particularly those responsible for binding of flavin. The results obtained are indicative of two opposite effects of viscous media with glycerol and sucrose: (1) destabilization of 4a-hydroperoxyflavin due to a change in the structural and dynamic properties of the protein and (2) stabilization of this intermediate by the decrease in the diffusion rate of its decay products.

Quantum Mechanics/Molecular Mechanics Simulations Show Saccharide Distortion is Required for Reaction in Hen Egg-White Lysozyme.

Hybrid quantum mechanics/molecular mechanics (QM/MM) calculations on lysozyme show significant distortion of the bound saccharide is required to facilitate the catalytic reaction.

A practical approach to steady-state kinetic analysis of cellulases acting on their natural insoluble substrate.

Measurement of steady-state rates (vSS) is straightforward in standard enzymology with soluble substrate, and it has been instrumental for comparative biochemical analyses within this area. For insoluble substrate, however, experimental values of vss remain controversial, and this has strongly limited the amount and quality of comparative analyses for cellulases and other enzymes that act on the surface of an insoluble substrate. In the current work, we have measured progress curves over a wide range of conditions for two cellulases, TrCel6A and TrCel7A from Trichoderma reesei, acting on their natural, insoluble substrate, cellulose. Based on this, we consider practical compromises for the determination of experimental vSS values, and propose a basic protocol that provides representative reaction rates and is experimentally simple so that larger groups of enzymes and conditions can be readily assayed with standard laboratory equipment. We surmise that the suggested experimental approach can be useful in comparative biochemical studies of cellulases; an area that remains poorly developed.

Exploring the chemistry and evolution of the isomerases.

Isomerization reactions are fundamental in biology, and isomers usually differ in their biological role and pharmacological effects. In this study, we have cataloged the isomerization reactions known to occur in biology using a combination of manual and computational approaches. This method provides a robust basis for comparison and clustering of the reactions into classes. Comparing our results with the Enzyme Commission (EC) classification, the standard approach to represent enzyme function on the basis of the overall chemistry of the catalyzed reaction, expands our understanding of the biochemistry of isomerization. The grouping of reactions involving stereoisomerism is straightforward with two distinct types (racemases/epimerases and cis-trans isomerases), but reactions entailing structural isomerism are diverse and challenging to classify using a hierarchical approach. This study provides an overview of which isomerases occur in nature, how we should describe and classify them, and their diversity.

In silico prediction of potential chemical reactions mediated by human enzymes.

BACKGROUND: Administered drugs are often converted into an ineffective or activated form by enzymes in our body. Conventional in silico prediction approaches focused on therapeutically important enzymes such as CYP450. However, there are more than thousands of different cellular enzymes that potentially convert administered drug into other forms. RESULT: We developed an in silico model to predict which of human enzymes including metabolic enzymes as well as CYP450 family can catalyze a given chemical compound. The prediction is based on the chemical and physical similarity between known enzyme substrates and a query chemical compound. Our in silico model was developed using multiple linear regression and the model showed high performance (AUC = 0.896) despite of the large number of enzymes. When evaluated on a test dataset, it also showed significantly high performance (AUC = 0.746). Interestingly, evaluation with literature data showed that our model can be used to predict not only enzymatic reactions but also drug conversion and enzyme inhibition. CONCLUSION: Our model was able to predict enzymatic reactions of a query molecule with a high accuracy. This may foster to discover new metabolic routes and to accelerate the computational development of drug candidates by enabling the prediction of the potential conversion of administered drugs into active or inactive forms.

Dual emissive bispyrene peptide probes for highly sensitive measurements of trypsin activity and evaluation of trypsin inhibitors.

Peptide substrates were double labeled with pyrenes to prepare fluorescent probes for highly sensitive detection of protease activity and evaluation of protease inhibitors using pyrene monomer/excimer signals. Two proximate pyrene moieties formed excited state dimers in the probes, and these pyrene excimer formations were dissociated by tryptic digestion. The specificity constant of the optimum bispyrene peptide probe was 2.7 times higher than that of the conventional peptide-4-methylcoumarin amide. Moreover, our probe had high sensitivity with an estimated detection limit for trypsin of 4.11 pM. The half maximal inhibitory concentration and dissociation constant of the Bowman-Birk inhibitor were successfully estimated.

Trend to Equilibrium for a Reaction-Diffusion System Modelling Reversible Enzyme Reaction.

A spatio-temporal evolution of chemicals appearing in a reversible enzyme reaction and modelled by a four-component reaction-diffusion system with the reaction terms obtained by the law of mass action is considered. The large time behaviour of the system is studied by means of entropy methods.

Unprecedented homotopy perturbation method for solving nonlinear equations in the enzymatic reaction of glucose in a spherical matrix.

The theory of glucose-responsive composite membranes for the planar diffusion and reaction process is extended to a microsphere membrane. The theoretical model of glucose oxidation and hydrogen peroxide production in the chitosan-aliginate microsphere has been discussed in this manuscript for the first time. We have successfully reported an analytical derived methodology utilizing homotopy perturbation to perform the numerical simulation. The influence and sensitive analysis of various parameters on the concentrations of gluconic acid and hydrogen peroxide are also discussed. The theoretical results enable to predict and optimize the performance of enzyme kinetics.

A Tandem Enzymatic sp2 -C-Methylation Process: Coupling in Situ S-Adenosyl-l-Methionine Formation with Methyl Transfer.

A one-pot, two-step biocatalytic platform for the regiospecfic C-methylation and C-ethylation of aromatic substrates is described. The tandem process utilises SalL (Salinospora tropica) for in situ synthesis of S-adenosyl-l-methionine (SAM), followed by alkylation of aromatic substrates by the C-methyltransferase NovO (Streptomyces spheroides). The application of this methodology is demonstrated for the regiospecific labelling of aromatic substrates by the transfer of methyl, ethyl and isotopically labelled 13 CH3,13 CD3 and CD3 groups from their corresponding SAM analogues formed in situ.

Type†III Polyketide Synthases: Functional Classification and Phylogenomics.

Polyketide synthases (PKSs) catalyze the sequential condensation of simple acetate units to produce a large class of natural products, including pharmacologically valuable compounds. PKSs are classified into three types on the basis of their domain structures; type III PKSs have the simplest domain structure, although their products have various structures and functions. The sequence-function relationship is fundamental for predicting enzyme functions, but it has not been well investigated in type III PKSs to date. Consequently, the current methods for predicting type III PKS functions are still immature in comparison with those that target type I/II PKSs. In this review we summarize the current functional and phylogenomic knowledge about type III PKSs and propose a new classification of their enzymatic reactions. We also discuss possible directions for the development of better computational tools for functional prediction of type III PKS homologues.

Elucidation of a carboxylate O-methyltransferase NcmP in nocamycin biosynthetic pathway.

Nocamycins belong to the tetramic acid family natural products and show potent antimicrobial activity. Recently, the biosynthetic gene cluster of nocamycin was identified from the rare actinomycete Saccharothrix syringae and an S-adenosylmethionine (SAM) dependent methyltransferase gene NcmP was found to be located within the gene cluster. In this report, the methyltransferase gene NcmP was disrupted and a new nocamycin intermediate nocamycin E was isolated from the mutant strain. Meanwhile, NcmP was heterologously expressed in Escherichia coli BL21 (DE3) and biochemically characterized as a carboxylate O-methyltransferase in nocamycin biosynthetic pathway. Compared to nocamycin I, nocamycin E showed inferior antibacterial activity, indicating the methyl group is essential to antibacterial activity.

ß-Glucuronidase-coupled assays of glucuronoyl esterases.

Glucuronoyl esterases (GEs) are microbial enzymes with potential to cleave the ester bonds between lignin alcohols and xylan-bound 4-O-methyl-d-glucuronic acid in plant cell walls. This activity renders GEs attractive research targets for biotechnological applications. One of the factors impeding the progress in GE research is the lack of suitable substrates. In this work, we report a facile preparation of methyl esters of chromogenic 4-nitrophenyl and 5-bromo-4-chloro-3-indolyl ß-D-glucuronides for qualitative and quantitative GE assay coupled with ß-glucuronidase as the auxiliary enzyme. The indolyl derivative affording a blue indigo-type product is suitable for rapid and sensitive assay of GE in commercial preparations as well as for high throughput screening of microorganisms and genomic and metagenomic libraries.

Novel fluorescent substrates for detection of trypsin activity and inhibitor screening by self-quenching.

A group of self-quenching-based substrates with two fluorescent peptides for detection of trypsin activity was designed and synthesized. The substrates could be easily synthesized using simple solid-phase peptide synthesis techniques. Two fluorescent peptide substrates for trypsin were conjugated to the amino groups of lysine as a branched unit. The fluorescence of these substrates was self-quenched owing to the highly assembled fluorophores on the substrates. The release of these concentrated fluorophores by proteases allows for fluorescence recovery. Self-quenching reduced the fluorescence of the substrates by 64.1%, and the fluorescence intensity was recovered by the release of the fluorophores from the substrate peptides via tryptic cleavage. The kinetic assay revealed that the kcat/Km values of the substrates were almost comparable to those of the standard fluorescent probe, peptide-MCA. The detection limit for trypsin was 111pM, and the calculation of IC50 and Ki values for the Bowman-Birk inhibitor was achieved using these substrates. These easily synthesizable self-quenching-based substrates have the potential to be useful for the detection of other disease-related protease activities.