Design of Deinococcus radiodurans thioredoxin reductase with altered thioredoxin specificity using computational alanine mutagenesis

In this study, the X-ray crystal structure of the complex between Escherichia coli thioredoxin reductase (EC TrxR) and its substrate thioredoxin (Trx) was used as a guide to design a Deinococcus radiodurans TrxR (DR TrxR) mutant with altered Trx specificity. Previous studies have shown that TrxRs ha...

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Bibliographic Details
Published in:Protein science 2011-06, Vol.20 (6), p.1021-1029
Main Authors: Obiero, Josiah, Sanders, David A R
Format: Article
Language:English
Subjects:
Alanine - genetics
Amino Acid Sequence
Bacteriology
Crystal structure
Crystallography, X-Ray
Deinococcus - enzymology
Deinococcus - genetics
Deinococcus radiodurans
Escherichia coli
Escherichia coli - enzymology
Models, Molecular
Molecular Sequence Data
Mutagenesis
Mutagenesis, Site-Directed
Point Mutation
Protein Engineering - methods
Sequence Alignment
Substrate Specificity
Thioredoxin-Disulfide Reductase - chemistry
Thioredoxin-Disulfide Reductase - genetics
Thioredoxin-Disulfide Reductase - metabolism
Thioredoxins - metabolism
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Summary:In this study, the X-ray crystal structure of the complex between Escherichia coli thioredoxin reductase (EC TrxR) and its substrate thioredoxin (Trx) was used as a guide to design a Deinococcus radiodurans TrxR (DR TrxR) mutant with altered Trx specificity. Previous studies have shown that TrxRs have higher affinity for cognate Trxs (same species) than that for Trxs from different species. Computational alanine scanning mutagenesis and visual inspection of the EC TrxR-Trx interface suggested that only four residues (F81, R130, F141, and F142) account for the majority of the EC TrxR-Trx interface stability. Individual replacement of equivalent residues in DR TrxR (M84, K137, F148, and F149) with alanine resulted in drastic changes in binding affinity, confirming that the four residues account for most of TrxR-Trx interface stability. When M84 and K137 were changed to match equivalent EC TrxR residues (K137R and M84F), the DR TrxR substrate specificity was altered from its own Trx to that of EC Trx. The results suggest that a small subset of the TrxR-Trx interface residues is responsible for the majority of Trx binding affinity and species-specific recognition.
ISSN:0961-8368
1469-896X
DOI:10.1002/pro.635