4350-09-8Relevant articles and documents
Synthesis of redox-active fluorinated 5-hydroxytryptophans as molecular reporters for biological electron transfer
Ohler, Amanda,Long, Hanna,Ohgo, Kei,Tyson, Kristin,Murray, David,Davis, Amanda,Whittington, Chris,Hvastkovs, Eli G.,Duffy, Liam,Haddy, Alice,Sargent, Andrew L.,Allen, William E.,Offenbacher, Adam R.
supporting information, p. 3107 - 3110 (2021/04/02)
Fluorinated 5-hydroxytryptophans (Fn-5HOWs) were synthesized in gram scale quantities and incorporated into a β-hairpin peptide and the protein azurin. The redox-active Fn-5HOWs exhibit unique radical spectroscopic signatures that expand the function of 5HOW as probes for biological electron transfer.
Mutagenesis of an Active-Site Loop in Tryptophan Hydroxylase Dramatically Slows the Formation of an Early Intermediate in Catalysis
Subedi, Bishnu P.,Fitzpatrick, Paul F.
supporting information, p. 5185 - 5192 (2018/04/23)
Solution studies of the aromatic amino acid hydroxylases are consistent with the FeIVO intermediate not forming until both the amino acid and tetrahydropterin substrates have bound. Structural studies have shown that the positions of active-site loops differs significantly between the free enzyme and the enzyme-amino acid-tetrahydropterin complex. In tryptophan hydroxylase (TrpH) these mobile loops contain residues 124-134 and 365-371, with a key interaction involving Ile366. The I366N mutation in TrpH results in decreases of 1-2 orders of magnitude in the kcat and kcat/Km values. Single turnover analyses establish that the limiting rate constant for turnover is product release for the wild-type enzyme but is formation of the first detectable intermediate I in catalysis in the mutant enzyme. The mutation does not alter the kinetics of NO binding to the ternary complex nor does it uncouple FeIVO formation from amino acid hydroxylation. The effects on the kcat value of wild-type TrpH of changing viscosity are consistent with rate-limiting product release. While the effect of viscosity on the kcat/KO2 value is small, consistent with reversible oxygen binding, the effects on the kcat/Km values for tryptophan and the tetrahydropterin are large, with the latter value exceeding the expected limit and varying with the identity of the viscogen. In contrast, the kinetic parameters of I366N TrpH show small changes with viscosity. The results are consistent with binding of the amino acid and pterin substrate to form the ternary complex being directly coupled to closure of loops over the active site and formation of the reactive complex. The mutation destabilizes this initial event.
A Panel of TrpB Biocatalysts Derived from Tryptophan Synthase through the Transfer of Mutations that Mimic Allosteric Activation
Murciano-Calles, Javier,Romney, David K.,Brinkmann-Chen, Sabine,Buller, Andrew R.,Arnold, Frances H.
supporting information, p. 11577 - 11581 (2016/10/24)
Naturally occurring enzyme homologues often display highly divergent activity with non-natural substrates. Exploiting this diversity with enzymes engineered for new or altered function, however, is laborious because the engineering must be replicated for each homologue. A small set of mutations of the tryptophan synthase β-subunit (TrpB) from Pyrococcus furiosus, which mimics the activation afforded by binding of the α-subunit, was demonstrated to have a similar activating effect in different TrpB homologues with as little as 57 % sequence identity. Kinetic and spectroscopic analyses indicate that the mutations function through the same mechanism: mimicry of α-subunit binding. From these enzymes, we identified a new TrpB catalyst that displays a remarkably broad activity profile in the synthesis of 5-substituted tryptophans. This demonstrates that allosteric activation can be recapitulated throughout a protein family to explore natural sequence diversity for desirable biocatalytic transformations.