1821-52-9Relevant articles and documents
Cloning and characterization of indolepyruvate decarboxylase from Methylobacterium extorquens AM1
Fedorov,Doronina,Trotsenko
, p. 1435 - 1443 (2010)
For the first time for methylotrophic bacteria an enzyme of phytohormone indole-3-acetic acid (IAA) biosynthesis, indole-3-pyruvate decarboxylase (EC 4.1.1.74), has been found. An open reading frame (ORF) was identified in the genome of facultative methylotroph Methylobacterium extorquens AM1 using BLAST. This ORF encodes thiamine diphosphate-dependent 2-keto acid decarboxylase and has similarity with indole-3-pyruvate decarboxylases, which are key enzymes of IAA biosynthesis. The ORF of the gene, named ipdC, was cloned into overexpression vector pET-22b(+). Recombinant enzyme IpdC was purified from Escherichia coli BL21(DE3) and characterized. The enzyme showed the highest k cat value for benzoylformate, albeit the indolepyruvate was decarboxylated with the highest catalytic efficiency (k cat/K m). The molecular mass of the holoenzyme determined using gel-permeation chromatography corresponds to a 245-kDa homotetramer. An ipdC-knockout mutant of M. extorquens grown in the presence of tryptophan had decreased IAA level (46% of wild type strain). Complementation of the mutation resulted in 6.3-fold increase of IAA concentration in the culture medium compared to that of the mutant strain. Thus involvement of IpdC in IAA biosynthesis in M. extorquens was shown.
Stepwise O-Atom Transfer in Heme-Based Tryptophan Dioxygenase: Role of Substrate Ammonium in Epoxide Ring Opening
Shin, Inchul,Ambler, Brett R.,Wherritt, Daniel,Griffith, Wendell P.,Maldonado, Amanda C.,Altman, Ryan A.,Liu, Aimin
supporting information, p. 4372 - 4379 (2018/04/05)
Heme-based tryptophan dioxygenases are established immunosuppressive metalloproteins with significant biomedical interest. Here, we synthesized two mechanistic probes to specifically test if the α-amino group of the substrate directly participates in a critical step of the O atom transfer during catalysis in human tryptophan 2,3-dioxygenase (TDO). Substitution of the nitrogen atom of the substrate to a carbon (probe 1) or oxygen (probe 2) slowed the catalytic step following the first O atom transfer such that transferring the second O atom becomes less likely to occur, although the dioxygenated products were observed with both probes. A monooxygenated product was also produced from probe 2 in a significant quantity. Analysis of this new product by HPLC coupled UV-vis spectroscopy, high-resolution mass spectrometry, 1H NMR, 13C NMR, HSQC, HMBC, and infrared (IR) spectroscopies concluded that this monooxygenated product is a furoindoline compound derived from an unstable epoxyindole intermediate. These results prove that small molecules can manipulate the stepwise O atom transfer reaction of TDO and provide a showcase for a tunable mechanism by synthetic compounds. The product analysis results corroborate the presence of a substrate-based epoxyindole intermediate during catalysis and provide the first substantial experimental evidence for the involvement of the substrate α-amino group in the epoxide ring-opening step during catalysis. This combined synthetic, biochemical, and biophysical study establishes the catalytic role of the α-amino group of the substrate during the O atom transfer reactions and thus represents a substantial advance to the mechanistic comprehension of the heme-based tryptophan dioxygenases.
Synthesis of All Nineteen Appropriately Protected Chiral α-Hydroxy Acid Equivalents of the α-Amino Acids for Boc Solid-Phase Depsi-Peptide Synthesis
Deechongkit, Songpon,You, Shu-Li,Kelly, Jeffery W.
, p. 497 - 500 (2007/10/03)
(Equation presented) The preparation of depsi-peptides, amide-to-ester-substituted peptides used to probe the role of hydrogen bonding in protein folding energetics, is accomplished by replacing specific L-α-amino acid residues by their α-hydroxy acid cou
