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Effect of mutation at valine 61 on the three-dimensional structure, stability, and redox potential of cytochrome b5.

To elucidate the role played by Val61 of cytochrome b(5), this residue of the tryptic fragment of bovine liver cytochrome b(5) was chosen for replacement with tyrosine (Val61Tyr), histidine (Val61His), glutamic acid (Val61Glu), and lysine (Val61Lys) by means of site-directed mutagenesis. The mutants Val61Tyr, Val61Glu, Val61His, and Val61Lys exhibit electronic spectra identical to that of the wild type, suggesting that mutation at Val61 did not affect the overall protein structure significantly. The redox potentials determined by differential pulse voltammetry were -10 (wild type), -25 (Val61Glu), -33 (Val61Tyr), 12 (Val61His), and 17 mV (Val61Lys) versus NHE. The thermal stabilities and urea-mediated denaturation of wild-type cytochrome b(5) and its mutants were in the following order: wild type > Val61Glu > Val61Tyr > Val61His > Val61Lys. The kinetics of denaturation of cytochrome b(5) by urea was also analyzed. The first-order rate constants of heme transfer between cytochrome b(5) and apomyoglobin at 20 +/- 0.2 degrees C were 0.25 +/- 0.01 (wild type), 0.42 +/- 0.02 (Val61Tyr), 0.93 +/- 0.04 (Val61Glu), 2.88 +/- 0.01 (Val61His), and 3.88 +/- 0.02 h(-)(1) (Val61Lys). The crystal structure of Val61His was determined using the molecular replacement method and refined at 2.1 A resolution, showing that the imidazole side chain of His61 points away from the heme-binding pocket and extends into the solvent, the coordination distances from Fe to NE2 atoms of two axial ligands are approximately 0.6 A longer than the reported value, and the hydrogen bond network involving Val61, the heme propionates, and three water molecules no longer exists. We conclude that the conserved residue Val61 is located at one of the key positions, the "electrostatic potential" around the heme-exposed area and the hydrophobicity of the heme pocket are determinant factors modulating the redox potential of cytochrome b(5), and the hydrogen bond network around the exposed heme edge is also an important factor affecting the heme stability.

Animals↗

Stereospecific NMR assignments of prochiral methyls, rotameric states and dynamics of valine residues in malate synthase G.

Near complete stereospecific assignments of the prochiral methyl carbons of Leu and Val residues in malate synthase G, a 723 residue enzyme, are reported. Assignments were obtained on the basis of a 10% fractional (13)C-labeling strategy developed by Wüthrich and co-workers [Neri, D; Szyperski, T; Otting, G; Senn, H; Wüthrich, K. Biochemistry 1989, 28, 7510-7516] and, in the case of Val residues, supplemented with results from a series of new methyl-TROSY quantitative J experiments for measuring (3)J(C)(gamma)(N) and (3)J(C)(gamma)(C)' couplings. The measured (3)J couplings were also used to probe Val side chain dynamics. A strong correlation is observed between rotamer averaging established on the basis of the couplings and side chain millisecond time scale dynamics measured using methyl-TROSY based (1)H-(13)C multiple quantum relaxation dispersion experiments.

Carbon Isotopes↗

Enzymatic synthesis of gamma-glutamylvaline to improve the bitter taste of valine.

The taste of several bitter amino acids is reduced, sourness produced, and preference increased by gamma-glutamylization. An enzymatic method for synthesizing gamma-Glu-Val involving bacterial gamma-glutamyltranspeptidase (GGT) was developed. The optimum reaction conditions for the synthesis of gamma-Glu-Val were 20 mM Gln, 300 mM Val, and 0.04 U/ml GGT, pH 10. After 3-hr incubation at 37 degrees C, 17.6 mM gamma-Glu-Val was obtained, with the yield being 88%. gamma-Glu-Val was purified on a Dowex 1 x 8 column and then identified by NMR.

Dipeptides↗

Valine pathway is more crucial than phenyl propanoid pathway in regulating capsaicin biosynthesis in Capsicum frutescens mill.

Capsaicin, a pungency factor alkaloid of Capsicum fruits, is biosynthesized by enzymatic condensation of vanillylamine, a phenyl propanoid intermediate, and 8-methyl-nonenoic acid, a fatty acid derivative from the leucine/valine pathway by capsaicin synthase. Biotic elicitors, such as aqueous mycelial extracts of Rhizopus oligosporus and Aspergillus niger, and abiotic elicitors, such as salicylic acid and methyl jasmonate, were incorporated to cell-suspension cultures of Capsicum frutescens. The maximum elicitation of capsaicinoids to the extent of 6-folds was noticed when mycelial extracts of R. oligosporus were incorporated to cell-suspension cultures. The phenyl propanoid intermediates were elicited to the extent of 3-4-fold, whereas the levels of 8-methyl-nonanoic acid increased 6-fold in R. oligosporus mycelial-extract-treated cultures. The enzymatic assays of caffeic acid O-methyl transferases, keto acyl synthase, and capsaicin synthase were performed in elicited and nonelicited cell-suspension cultures. These enzymes were overexpressed in R. oligosporus mycelial-extract-treated suspension cultures, which was also confirmed by reverse transcriptase polymerase chain reaction studies. This is the first paper on the overexpression of 8-methyl-nonanoic acid leading to capsaicin biosynthesis using elicitors.

Benzylamines↗