Insights into the mechanism and substrate specificity of human lysine -specific demethylase-1

Histones are small basic proteins that function to organize DNA in cells. The nucleosomal core particle, the fundamental unit of chromatin, consists of 146-147 base pairs of DNA wound around a complex of two H2A-H2B histone dimers and an H3-H4 tetramer. Histones are subject to a myriad of post-translational modifications, including methylation, phosphorylation, acetylation, and ubiquitination, which function in the regulation of various cell processes, including gene transcription. Lysine-Specific Demethylase-1 (LSD1), a member of the monoamine oxidase (MAO) family of flavoprotein amine oxidases, has been shown to remove methyl groups from lysine residues four and nine of histone H3 (H3K4 and H3K9), as well as lysine 370 of the tumor suppressor protein p53. The enzyme has been shown to play a role in controlling cell differentiation, and its expression correlates with neuroblastoma and prostate cancer progression; thus, selective inhibitors of LSD1 may be beneficial for treatment of various diseases. Thorough characterization of the mechanism and substrate specificity of LSD1 are essential for the development of such inhibitors, as well as for increasing the understanding of transcriptional regulation and cancer proliferation. The substrate specificity of LSD1 has been studied in vitro by utilizing a series of peptide substrates corresponding to the amino acid sequence of the N-terminal tail of histone H3. Recombinant human LSD1 was shown to act specifically on the dimethylated H3K4 residue in vitro, with arginine residues in the peptide substrate being essential for recognition of the substrate by the enzyme. Steady-state and transient kinetic studies have shown that C-H bond cleavage is rate-limiting in oxidation of a peptide substrate by LSD1. Furthermore, the redox potential of LSD1, significantly higher than that of free flavin, demonstrates that the enzyme provides a more favorable environment for flavin-catalyzed oxidation. Finally, evidence suggests that a lysine residue conserved in the MAO family, Lys661 in LSD1, may play a role in enzyme stability. These studies contribute to the overall understanding of the mechanism and substrate-specificity of LSD1

Insights into the mechanism and substrate specificity of human lysine -specific demethylase-1

Histones are small basic proteins that function to organize DNA in cells. The nucleosomal core particle, the fundamental unit of chromatin, consists of 146-147 base pairs of DNA wound around a complex of two H2A-H2B histone dimers and an H3-H4 tetramer. Histones are subject to a myriad of post-translational modifications, including methylation, phosphorylation, acetylation, and ubiquitination, which function in the regulation of various cell processes, including gene transcription. Lysine-Specific Demethylase-1 (LSD1), a member of the monoamine oxidase (MAO) family of flavoprotein amine oxidases, has been shown to remove methyl groups from lysine residues four and nine of histone H3 (H3K4 and H3K9), as well as lysine 370 of the tumor suppressor protein p53. The enzyme has been shown to play a role in controlling cell differentiation, and its expression correlates with neuroblastoma and prostate cancer progression; thus, selective inhibitors of LSD1 may be beneficial for treatment of various diseases. Thorough characterization of the mechanism and substrate specificity of LSD1 are essential for the development of such inhibitors, as well as for increasing the understanding of transcriptional regulation and cancer proliferation. The substrate specificity of LSD1 has been studied in vitro by utilizing a series of peptide substrates corresponding to the amino acid sequence of the N-terminal tail of histone H3. Recombinant human LSD1 was shown to act specifically on the dimethylated H3K4 residue in vitro, with arginine residues in the peptide substrate being essential for recognition of the substrate by the enzyme. Steady-state and transient kinetic studies have shown that C-H bond cleavage is rate-limiting in oxidation of a peptide substrate by LSD1. Furthermore, the redox potential of LSD1, significantly higher than that of free flavin, demonstrates that the enzyme provides a more favorable environment for flavin-catalyzed oxidation. Finally, evidence suggests that a lysine residue conserved in the MAO family, Lys661 in LSD1, may play a role in enzyme stability. These studies contribute to the overall understanding of the mechanism and substrate-specificity of LSD1

Isotope Effects Suggest a Stepwise Mechanism for Berberine Bridge Enzyme

The flavoprotein Berberine Bridge Enzyme (BBE) catalyzes the regioselective oxidative cyclization of (<i>S</i>)-reticuline to (<i>S</i>)-scoulerine in an alkaloid biosynthetic pathway. A series of solvent and substrate deuterium kinetic isotope effect studies were conducted to discriminate between a concerted mechanism, in which deprotonation of the substrate phenol occurs before or during the transfer of a hydride from the substrate to the flavin cofactor and substrate cyclization, and a stepwise mechanism, in which hydride transfer results in the formation of a methylene iminium ion intermediate that is subsequently cyclized. The substrate deuterium isotope effect of 3.5 on <i>k</i>_red, the rate constant for flavin reduction, is pH-independent, indicating that C–H bond cleavage is rate-limiting during flavin reduction. Solvent isotope effects on <i>k</i>_red are equal to 1 for both wild-type BBE and the E417Q mutant, indicating that solvent exchangeable protons are not in flight during or before flavin reduction, thus eliminating a fully concerted mechanism as a possibility for catalysis by BBE. An intermediate was not detected by rapid chemical quench or continuous-flow mass spectrometry experiments, indicating that it must be short-lived

Structure of the Flavoprotein Tryptophan 2‑Monooxygenase, a Key Enzyme in the Formation of Galls in Plants

The flavoprotein tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to yield indole-3-acetamide. This is the initial step in the biosynthesis of the plant growth hormone indole-acetic acid by bacterial pathogens that cause crown gall and related diseases. The structure of the enzyme from <i>Pseudomonas savastanoi</i> has been determined by X-ray diffraction methods to a resolution of 1.95 Å. The overall structure of the protein shows that it has the same fold as members of the monoamine oxidase family of flavoproteins, with the greatest similarities to the l-amino acid oxidases. The location of bound indole-3-acetamide in the active site allows identification of residues responsible for substrate binding and specificity. Two residues in the enzyme are conserved in all members of the monoamine oxidase family, Lys365 and Trp466. The K365M mutation decreases the <i>k</i>_cat and <i>k</i>_cat/<i>K</i>_Trp values by 60000- and 2 million-fold, respectively. The deuterium kinetic isotope effect increases to 3.2, consistent with carbon–hydrogen bond cleavage becoming rate-limiting in the mutant enzyme. The W466F mutation decreases the <i>k</i>_cat value <2-fold and the <i>k</i>_cat/<i>K</i>_Trp value only 5-fold, while the W466M mutation results in an enzyme lacking flavin and detectable activity. This is consistent with a role for Trp466 in maintaining the structure of the flavin-binding site in the more conserved FAD domain