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Fig. 4 in Site directed mutagenesis of Catharanthus roseus (+)-vincadifformine 19-hydroxylase (CYP71BY3) results in two distinct enzymatic functions

Fig. 4. GOLD's highest rated poses for (¡)-tabersonine and (þ)-vincadifformine into the V19H model generated from the 3ruk template with and without increased flexibility in the binding site residues. ()-tabersonine (B) and (+)-vincadifformine (A) best poses are shown in a rigid binding site (top) and a flexible binding site (bottom). The 19C is labelled in both ligands (arrows), and neither ligand is docked in the correct orientation for oxidation in a rigid binding site (top. However, in a flexible binding site, (+)-vincadifformine is in the correct orientation for 19C oxidation (bottom; B), whereas ()-tabersonine is not (bottom; A). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5 in Site directed mutagenesis of Catharanthus roseus (+)-vincadifformine 19-hydroxylase (CYP71BY3) results in two distinct enzymatic functions

Fig. 5. The four residues in the V19H (left) and T3O (right) binding sites that were predicted to affect the opposite enantioselectivity of these highly homologous enzymes using models created on the 3ruk template. Residues shown in the V19H pocket are K106, S312, A376, and F377. Residues shown in the T3O pocket are R105, T311, P375, and L377. The model generated for T19H (3ruk template) looks identical to the T3O pocket, and the correlating residues are R101, T310, P374, and L375.

Fig. 3 in Site directed mutagenesis of Catharanthus roseus (+)-vincadifformine 19-hydroxylase (CYP71BY3) results in two distinct enzymatic functions

Fig. 3. Saturation kinetics of V19H: comparative (þ)-vincadifformine saturation curve in the absence or presence of (¡)-vincadifformine. The rate of (+)-vincadifformine consumption by V19H was reduced in the presence of 3 μM of ()-vincadifformine within the range of 15–20 μM of substrate yet remains unchanged at low and saturated concentrations. Error bars indicate the standard deviation from three technical replicate assays.

Fig. 7. The V19H four-point mutant coupled with T3R in Site directed mutagenesis of Catharanthus roseus (+)-vincadifformine 19-hydroxylase (CYP71BY3) results in two distinct enzymatic functions

Fig. 7. The V19H four-point mutant coupled with T3R converts (¡)-tabersonine to 2,3-dihydro-3-hydroxytabersonine. Traces from in vitro experiments with ()-tabersonine (1) and V19H four-point mutant microsomes or T3O microsomes in the absence or presence of purified recombinant T3R are shown. Yeast microsomes containing the V19H 4-point mutant (red) (V19HL106R–S312T-A376P–F377L) convert ()-tabersonine to tabersonine 2,3-epoxides (5) in the absence of T3R, and to 2,3-dihydro-3-hydroxytabersonine in the presence of T3R. Yeast microsomes containing wild-type T3O (blue) were used as positive controls for tabersonine-2,3-epoxide (5) and 2,3-dihydro-3-hydroxytabersonine biosynthesis in the absence and presence of T3R, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2 in Site directed mutagenesis of Catharanthus roseus (+)-vincadifformine 19-hydroxylase (CYP71BY3) results in two distinct enzymatic functions

Fig. 2. Representation of the 3ruk V19H model superimposed onto the 3ruk template and the location of the catalytic heme. V19H model (purple) is shown in purple, and the 3ruk template is shown in pink with the heme binding site in yellow. The H-bond interaction (green) between PHE443 and CYS450, the first and eighth residues of the heme binding site, which is responsible for the beta bulge (yellow) around the heme cofactor (black). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 1 in Site directed mutagenesis of Catharanthus roseus (+)-vincadifformine 19-hydroxylase (CYP71BY3) results in two distinct enzymatic functions

Fig. 1. The formation of (þ)- and (¡)-aspidosperma MIAs in Catharanthus roseus. In multiple C. roseus tissues, the ()-aspidosperma pathways begin with the biosynthesis of ()-tabersonine (1) from O-acetylstemmadenine (18) through acetylstemmadenine oxygenase (ASO), geissoschizine synthase (GS), and hydrolase 1 (HL1), whereas the (+)-aspidosperma pathway requires hydrolase 3 or 4 (HL3/4) to produce (+)-vincadifformine (15). Leaf-specific enzymes (black) convert ()-tabersonine (1) to vindoline (6), which accumulates in the leaves, while root-specific enzymes (purple) are responsible for the formation of two major root alkaloids, lochnericine (7) and h¨orhammericine (8). A third major root alkaloid, (+)-echitovenine (17) is derived from (+)-vincadifformine (15) via (+)-minovincinine (16) by separate root-specific (blue) enzymes. The remaining ()-aspidosperma alkaloids shown are derived chemically (grey box) from ()-tabersonine (1) to generate ()-vincadifformine (12) or enzymatic conversion by T19H to form 19-hydroxytabersonine (10) or its 19-O acetyltabersonine (11) by the action of TAT. These MIAs [()-vincadifformine (12), 19-hydroxytabersonine (10) and 19-O acetyltabersonine (11)] do not naturally accumulate in C. roseus. T16H: tabersonine 16-hydroxylase [CYP71D12 (T16H1) or CYP71D351 (T16H2); 16OMT: tabersonine 16-O-methyltransferase; T3O: tabersonine 3-oxygenase (CYP71D1V2); T3R: tabersonine 3-reductase; NMT: N-methyltransferase; D4H: desacetoxyvindoline 4-hydroxylase; DAT: deacetylvindoline O-acetyltransferase; TEX: tabersonine epoxidase [CYP71D521 (TEX1) or CYP71D347 (TEX2)]; T19H: tabersonine 19-hydroxylase (CYP71BJ1); TAT: tabersonine 19-acetyltranferase; V19H: (+)-vincadifformine 19-hydroxylase (CYP71BY3); MAT: minovincinine 19-O-acetyltransferase. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

opencc-by-4.0Aug 2023View details →

A Method for Differentiating Sepsis and Septic Shock Based on Phenylalanine Hydroxylase and Lactate Dehydrogenase Activity

The present method relates to improving the prognosis, diagnosis, and treatment of sepsis and septic shock. Despite the availability of advanced treatment, sepsis, and septic shock have the highest mortality in the intensive care unit (ICU). Theories suggested that hyperinflammation is a major characteristic of septic shock, but oxidative stress plays a major role in disease pathogenesis. Nuclear Magnetic Resonance (NMR) spectroscopy-based quantitative assessment of metabolites was performed to compare the activity of lactate dehydrogenase and phenylalanine hydroxylase between sepsis, septic shock, and disease control in sepsis and septic shock by comparing pyruvate/lactate (Pyr/Lac) and phenylalanine/tyrosine (Phe/Tyr) ratios. These ratios were evaluated and found to be used as an effective tool for diagnosis, prognosis, evaluation of disease activity, and treatment response.

Fig. 5 in L-DOPA synthesis in Mucuna pruriens (L.) DC. is regulated by polyphenol oxidase and not CYP 450/tyrosine hydroxylase: An analysis of metabolic pathway using biochemical and molecular markers

Fig. 5. The amplicons generated using degenerate primer approach. (a) Lane M-DNA ladder, Lane 1–250 bp amplicon generated using MTH –F and MTH-R primer pairs of TH gene (b) Lane M-DNA ladder, Lane 1 and 2–800 bp amplicon using primers deduced from the peptide sequence derived through LCMS/MS.

Fig. 4 in L-DOPA synthesis in Mucuna pruriens (L.) DC. is regulated by polyphenol oxidase and not CYP 450/tyrosine hydroxylase: An analysis of metabolic pathway using biochemical and molecular markers

Fig. 4. Effect of enzyme inhibitors on L-DOPA production in callus cultures of M. pruriens was estimated using HPTLC. The culture without inhibitor was treated as negative control and cultures with different concentration of the inhibitor were the test samples. (Control-untreated, C = Cimetidine at 1.98 μM and 19.8 μM; Q = Quinidine at 1.46 μM and 14.6 μM; A = L-ascorbic acid at 567 μM and 851 μM; K = Kojic acid at 703 μM and 1055 μM).

Fig. 3 in L-DOPA synthesis in Mucuna pruriens (L.) DC. is regulated by polyphenol oxidase and not CYP 450/tyrosine hydroxylase: An analysis of metabolic pathway using biochemical and molecular markers

Fig. 3. Effect of substrate concentration on partially purified enzymes. The assay was performed for PPO activity with 50 mM catechol as substrate at pH 6.0 while keeping the temperature for reaction at 30 ◦ C. For TH activity, 30 mM L-tyrosine was the substrate and assay done at pH 7.0 and 25 ◦ C.

Fig. 2 in L-DOPA synthesis in Mucuna pruriens (L.) DC. is regulated by polyphenol oxidase and not CYP 450/tyrosine hydroxylase: An analysis of metabolic pathway using biochemical and molecular markers

Fig. 2. Effect of pH on the activity of partially purified enzymes from Mucuna pruriens. The assay was performed using 50 mM catechol and 30 mM L-tyrosine as substrates for the PPO and TH enzyme activity, respectively. Four different buffers with their optimal buffering capacity in the pH range of 3–10 were used in separate assays.

Fig. 7 in L-DOPA synthesis in Mucuna pruriens (L.) DC. is regulated by polyphenol oxidase and not CYP 450/tyrosine hydroxylase: An analysis of metabolic pathway using biochemical and molecular markers

Fig. 7. Homology modelling and secondary structure prediction of PPO enzyme from Mucuna pruriens (a) Predicted secondary structure of PPO (b) Phyre2 protein model for PPO with 3D model dimensions (in Å) (X:49.941 Y:64.463 Z:57.979). Image colored by rainbow N → C terminus (c) Three dimensional SWISS protein model for PPO enzyme with two active copper binding ligands (copper ions bridging oxygen moiety is illustrated as small yellow spheres highlighted in the box), conserved histidine residues and metal complex interactions (in dotted lines). Chain A for Ligand 1: H.183, H.204, H.213, F.367, H.371; metal interactions: A:H.183, A:H.204, A:H.213. Chain A for Ligand 2: H.337, H.341, F.367, H.370, H.371; metal interactions: A:H.337, A:H.341, A:H.371). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6. The 1800 in L-DOPA synthesis in Mucuna pruriens (L.) DC. is regulated by polyphenol oxidase and not CYP 450/tyrosine hydroxylase: An analysis of metabolic pathway using biochemical and molecular markers

Fig. 6. The 1800 bp amplicon of full-length PPO cDNA obtained after deducing the 5′and 3′ ends through RACE analysis. Lane 1- 1 Kb DNA marker, Lane 2 and 3 the amplicon in duplicate after amplification using gene specific primers.