Thioredoxin reductase - Thioredoxin fusion enzyme from Mycobacterium leprae: Comparison with the separately expressed thioredoxin reductase

Pan Fen Wang, Jovita Marcinkeviciene, Charles H. Williams, John S. Blanchard

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin (Trx) by NADPH. A unique gene organization of TrxR and Trx has been found in Mycobacterium leprae, where TrxR and Trx are encoded by a single gene and, therefore, are expressed as a fusion protein (MlTrxR-Trx). This fusion enzyme is able to catalyze the reduction of thioredoxin or 5,5'-dithiobis(2- nitrobenzoic acid) or 1,4-naphthoquinone by NADPH, though the activity is much lower than that of Escherichia coli TrxR. It has been proposed that a large conformational change is required in catalysis of E. coli TrxR. Because the reductase portion of the enzyme from M. leprae shows significant primary structure similarity with E. coli TrxR, it is possible that MlTrxR-Trx may require a similar conformational change and that the change in conformation may be affected by the tethered Trx. The reductase has been expressed without Trx attached (MlTrxR). As reported here, comparison of the steady-state and pre-steady-state kinetics of MlTrxR-Trx with those of MlTrxR suggests that the low reductase activity of the fusion enzyme is an inherent property of the reductase, and that any steric limitation caused by the attached thioredoxin in the fusion protein makes only a minor contribution to the low activity. Titration of MlTrxR-Trx and MlTrxR with 3-aminopyridine adenine dinucleotide phosphate (AADP+), an NADP(H) analogue, results in only slight quenching of FAD fluorescence, suggesting an enzyme conformation in which the binding site of AADP+ is not close to the FAD, as in one of the conformations of E. coli TrxR.

Original languageEnglish (US)
Pages (from-to)16378-16389
Number of pages12
JournalBiochemistry
Volume37
Issue number46
DOIs
StatePublished - Nov 17 1998

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Thioredoxin-Disulfide Reductase
Mycobacterium leprae
Thioredoxins
Fusion reactions
Enzymes
Escherichia coli
Oxidoreductases
NADP
Conformations
Flavin-Adenine Dinucleotide
Genes
Dithionitrobenzoic Acid
Catalysis
Titration
Quenching
Proteins
Fluorescence

ASJC Scopus subject areas

  • Biochemistry

Cite this

Thioredoxin reductase - Thioredoxin fusion enzyme from Mycobacterium leprae : Comparison with the separately expressed thioredoxin reductase. / Wang, Pan Fen; Marcinkeviciene, Jovita; Williams, Charles H.; Blanchard, John S.

In: Biochemistry, Vol. 37, No. 46, 17.11.1998, p. 16378-16389.

Research output: Contribution to journalArticle

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abstract = "Thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin (Trx) by NADPH. A unique gene organization of TrxR and Trx has been found in Mycobacterium leprae, where TrxR and Trx are encoded by a single gene and, therefore, are expressed as a fusion protein (MlTrxR-Trx). This fusion enzyme is able to catalyze the reduction of thioredoxin or 5,5'-dithiobis(2- nitrobenzoic acid) or 1,4-naphthoquinone by NADPH, though the activity is much lower than that of Escherichia coli TrxR. It has been proposed that a large conformational change is required in catalysis of E. coli TrxR. Because the reductase portion of the enzyme from M. leprae shows significant primary structure similarity with E. coli TrxR, it is possible that MlTrxR-Trx may require a similar conformational change and that the change in conformation may be affected by the tethered Trx. The reductase has been expressed without Trx attached (MlTrxR). As reported here, comparison of the steady-state and pre-steady-state kinetics of MlTrxR-Trx with those of MlTrxR suggests that the low reductase activity of the fusion enzyme is an inherent property of the reductase, and that any steric limitation caused by the attached thioredoxin in the fusion protein makes only a minor contribution to the low activity. Titration of MlTrxR-Trx and MlTrxR with 3-aminopyridine adenine dinucleotide phosphate (AADP+), an NADP(H) analogue, results in only slight quenching of FAD fluorescence, suggesting an enzyme conformation in which the binding site of AADP+ is not close to the FAD, as in one of the conformations of E. coli TrxR.",
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