Mutation of cysteine residues alters the heme-binding pocket of indoleamine 2,3-dioxygenase-1

Christopher J D Austin, Priambudi Kosim-Satyaputra, Jason R. Smith, Robert D. Willows, Joanne F. Jamie

Research output: Contribution to journalArticleResearchpeer-review

Abstract

The hemoprotein indoleamine 2,3-dioxygenase-1 (IDO1) is the first and rate-limiting enzyme in mammalian tryptophan metabolism. Interest in IDO1 continues to grow, due to the ever expanding influence IDO1 plays in the immune response. This study examined the contribution of all individual cysteine residues towards the overall catalytic properties and stability of recombinant human IDO1 via mutagenesis studies using a range of biochemical and spectroscopic techniques, including in vitro kinetic assessment, secondary structure identification via circular dichroism spectroscopy and thermal stability assessment. Upon mutation of cysteine residues we observed changes in secondary structure (principally, shifting from α-helix/β-sheet features to random coil structures) that produced out of plane heme torsion and puckering, changes to thermal stability (including gains in stability for one mutant protein) and differences in enzymatic activity (such as, increased ability to convert non-natural substrates, e.g.d-tryptophan) from wild type IDO1 enzyme.

LanguageEnglish
Pages595-600
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume436
Issue number4
DOIs
Publication statusPublished - 12 Jul 2013

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Indoleamine-Pyrrole 2,3,-Dioxygenase
Heme
Cysteine
Mutation
Tryptophan
Thermodynamic stability
Hot Temperature
Circular dichroism spectroscopy
Mutagenesis
Mutant Proteins
Enzymes
Circular Dichroism
Metabolism
Torsional stress
Spectrum Analysis
Kinetics
Substrates

Cite this

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title = "Mutation of cysteine residues alters the heme-binding pocket of indoleamine 2,3-dioxygenase-1",
abstract = "The hemoprotein indoleamine 2,3-dioxygenase-1 (IDO1) is the first and rate-limiting enzyme in mammalian tryptophan metabolism. Interest in IDO1 continues to grow, due to the ever expanding influence IDO1 plays in the immune response. This study examined the contribution of all individual cysteine residues towards the overall catalytic properties and stability of recombinant human IDO1 via mutagenesis studies using a range of biochemical and spectroscopic techniques, including in vitro kinetic assessment, secondary structure identification via circular dichroism spectroscopy and thermal stability assessment. Upon mutation of cysteine residues we observed changes in secondary structure (principally, shifting from α-helix/β-sheet features to random coil structures) that produced out of plane heme torsion and puckering, changes to thermal stability (including gains in stability for one mutant protein) and differences in enzymatic activity (such as, increased ability to convert non-natural substrates, e.g.d-tryptophan) from wild type IDO1 enzyme.",
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Mutation of cysteine residues alters the heme-binding pocket of indoleamine 2,3-dioxygenase-1. / Austin, Christopher J D; Kosim-Satyaputra, Priambudi; Smith, Jason R.; Willows, Robert D.; Jamie, Joanne F.

In: Biochemical and Biophysical Research Communications, Vol. 436, No. 4, 12.07.2013, p. 595-600.

Research output: Contribution to journalArticleResearchpeer-review

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AB - The hemoprotein indoleamine 2,3-dioxygenase-1 (IDO1) is the first and rate-limiting enzyme in mammalian tryptophan metabolism. Interest in IDO1 continues to grow, due to the ever expanding influence IDO1 plays in the immune response. This study examined the contribution of all individual cysteine residues towards the overall catalytic properties and stability of recombinant human IDO1 via mutagenesis studies using a range of biochemical and spectroscopic techniques, including in vitro kinetic assessment, secondary structure identification via circular dichroism spectroscopy and thermal stability assessment. Upon mutation of cysteine residues we observed changes in secondary structure (principally, shifting from α-helix/β-sheet features to random coil structures) that produced out of plane heme torsion and puckering, changes to thermal stability (including gains in stability for one mutant protein) and differences in enzymatic activity (such as, increased ability to convert non-natural substrates, e.g.d-tryptophan) from wild type IDO1 enzyme.

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