Site‐directed mutagenesis of Glu477 of the human thioredoxin reductase (see figure) to glutamine, alanine, or lysine led to a significant drop in enzymatic activity. This study reinforces previous theoretical calculations which suggested that a swapping catalytic triad exists in the active site of this enzyme.

Site‐directed mutagenesis of Glu477 of the human thioredoxin reductase (see figure) to glutamine, alanine, or lysine led to a significant drop in enzymatic activity. This study reinforces previous theoretical calculations which suggested that a swapping catalytic triad exists in the active site of this enzyme.

Thioredoxin reductases catalyse the reduction of thioredoxin disulfide and some other oxidised cell constituents. They are homodimeric proteins containing one FAD and accepting one NADPH per subunit as essential cofactors. Some of these reductases contain a selenocysteine at the C terminus. Based on the X‐ray structure of rat thioredoxin reductase, homology models of human thioredoxin reductase were created and subsequently docked to thioredoxin to model the active complex. The formation of a new type of a catalytic triad between selenocysteine, histidine and a glutamate could be detected in the protein structure. By means of DFT (B3LYP, lacv3p**) calculations, we could show that the formation of such a triad is essential to support the proton transfer from selenol to a histidine to stabilise a selenolate anion, which is able to interact with the disulfide of thioredoxin and catalyses the reductive disulfide opening. Whereas a simple proton transfer from selenocysteine to histidine is thermodynamically disfavoured by some 18 kcal mol −1 , it becomes favoured when the carboxylic acid group of a glutamate stabilises the formed imidazole cation. An identical process with a cysteine instead of selenocysteine will require 4 kcal mol −1 more energy, which corresponds to a calculated equilibrium shift of ∼1000:1 or a 10 3 rate acceleration: a value close to the experimental one of about 10 2 times. These results give new insights into the catalytic mechanism of thioredoxin reductase and, for the first time, explain the advantage of the incorporation of a selenocysteine instead of a cysteine residue in a protein.

Thioredoxin reductases catalyse the reduction of thioredoxin disulfide and some other oxidised cell constituents. They are homodimeric proteins containing one FAD and accepting one NADPH per subunit as essential cofactors. Some of these reductases contain a selenocysteine at the C terminus. Based on the X‐ray structure of rat thioredoxin reductase, homology models of human thioredoxin reductase were created and subsequently docked to thioredoxin to model the active complex. The formation of a new type of a catalytic triad between selenocysteine, histidine and a glutamate could be detected in the protein structure. By means of DFT (B3LYP, lacv3p**) calculations, we could show that the formation of such a triad is essential to support the proton transfer from selenol to a histidine to stabilise a selenolate anion, which is able to interact with the disulfide of thioredoxin and catalyses the reductive disulfide opening. Whereas a simple proton transfer from selenocysteine to histidine is thermodynamically disfavoured by some 18 kcal mol −1 , it becomes favoured when the carboxylic acid group of a glutamate stabilises the formed imidazole cation. An identical process with a cysteine instead of selenocysteine will require 4 kcal mol −1 more energy, which corresponds to a calculated equilibrium shift of ∼1000:1 or a 10 3 rate acceleration: a value close to the experimental one of about 10 2 times. These results give new insights into the catalytic mechanism of thioredoxin reductase and, for the first time, explain the advantage of the incorporation of a selenocysteine instead of a cysteine residue in a protein.

Thioredoxin reductases catalyse the reduction of thioredoxin disulfide and some other oxidised cell constituents. They are homodimeric proteins containing one FAD and accepting one NADPH per subunit as essential cofactors. Some of these reductases contain a selenocysteine at the C terminus. Based on the X‐ray structure of rat thioredoxin reductase, homology models of human thioredoxin reductase were created and subsequently docked to thioredoxin to model the active complex. The formation of a new type of a catalytic triad between selenocysteine, histidine and a glutamate could be detected in the protein structure. By means of DFT (B3LYP, lacv3p**) calculations, we could show that the formation of such a triad is essential to support the proton transfer from selenol to a histidine to stabilise a selenolate anion, which is able to interact with the disulfide of thioredoxin and catalyses the reductive disulfide opening. Whereas a simple proton transfer from selenocysteine to histidine is thermodynamically disfavoured by some 18 kcal mol −1 , it becomes favoured when the carboxylic acid group of a glutamate stabilises the formed imidazole cation. An identical process with a cysteine instead of selenocysteine will require 4 kcal mol −1 more energy, which corresponds to a calculated equilibrium shift of ∼1000:1 or a 10 3 rate acceleration: a value close to the experimental one of about 10 2 times. These results give new insights into the catalytic mechanism of thioredoxin reductase and, for the first time, explain the advantage of the incorporation of a selenocysteine instead of a cysteine residue in a protein.

Experimental and theoretical investigations concerning the second‐to‐last step of the DXP/MEP pathway in isoprenoid biosynthesis in plants are reported. The proposed intrinsic or late intermediates 4‐oxo‐DMAPP ( 12 ) and 4‐hydroxy‐DMAPP ( 11 ) were synthesized in deuterium‐ or tritium‐labeled form according to new protocols especially adapted to work without protection of the diphosphate moiety. When the labeled compounds MEcPP ( 7 ), 11 , and 12 were applied to chromoplast cultures, aldehyde 12 was not incorporated. This finding is in agreement with a mechanistic and structural model of the responsible enzyme family: a three‐dimensional model of the fragment L271–A375 of the enzyme GcpE of Streptomyces coelicolor including NADPH, the Fe 4 S 4 cluster, and MEcPP ( 7 ) as ligand has been developed based on homology modeling techniques. The model has been accepted by the Protein Data Bank (entry code 1OX2). Supported by this model, semiempirical PM3 calculations were performed to analyze the likely catalysis mechanism of the reductive ring opening of MEcPP ( 7 ), hydroxyl abstraction, and formation of HMBPP ( 8 ). The mechanism is characterized by a proton transfer (presumably from a conserved arginine 286) to the substrate, accompanied by a ring opening without high energy barriers, followed by the transfer of two electrons delivered from the Fe 4 S 4 cluster, and finally proton transfer from a carboxylic acid side chain to the hydroxyl group to be removed from the ligand as water. The proposed mechanism is in agreement with all known experimental findings and the arrangement of the ligand within the enzyme. Thus, a very likely mechanism for the second to last step of the DXP/MEP pathway in isoprenoid biosynthesis in plants is presented. A principally similar mechanism is also expected for the reductive dehydroxylation of HMBPP ( 8 ) to IPP ( 9 ) and DMAPP ( 10 ) in the last step.

Experimental and theoretical investigations concerning the second‐to‐last step of the DXP/MEP pathway in isoprenoid biosynthesis in plants are reported. The proposed intrinsic or late intermediates 4‐oxo‐DMAPP ( 12 ) and 4‐hydroxy‐DMAPP ( 11 ) were synthesized in deuterium‐ or tritium‐labeled form according to new protocols especially adapted to work without protection of the diphosphate moiety. When the labeled compounds MEcPP ( 7 ), 11 , and 12 were applied to chromoplast cultures, aldehyde 12 was not incorporated. This finding is in agreement with a mechanistic and structural model of the responsible enzyme family: a three‐dimensional model of the fragment L271–A375 of the enzyme GcpE of Streptomyces coelicolor including NADPH, the Fe 4 S 4 cluster, and MEcPP ( 7 ) as ligand has been developed based on homology modeling techniques. The model has been accepted by the Protein Data Bank (entry code 1OX2). Supported by this model, semiempirical PM3 calculations were performed to analyze the likely catalysis mechanism of the reductive ring opening of MEcPP ( 7 ), hydroxyl abstraction, and formation of HMBPP ( 8 ). The mechanism is characterized by a proton transfer (presumably from a conserved arginine 286) to the substrate, accompanied by a ring opening without high energy barriers, followed by the transfer of two electrons delivered from the Fe 4 S 4 cluster, and finally proton transfer from a carboxylic acid side chain to the hydroxyl group to be removed from the ligand as water. The proposed mechanism is in agreement with all known experimental findings and the arrangement of the ligand within the enzyme. Thus, a very likely mechanism for the second to last step of the DXP/MEP pathway in isoprenoid biosynthesis in plants is presented. A principally similar mechanism is also expected for the reductive dehydroxylation of HMBPP ( 8 ) to IPP ( 9 ) and DMAPP ( 10 ) in the last step.

Experimental and theoretical investigations concerning the second‐to‐last step of the DXP/MEP pathway in isoprenoid biosynthesis in plants are reported. The proposed intrinsic or late intermediates 4‐oxo‐DMAPP ( 12 ) and 4‐hydroxy‐DMAPP ( 11 ) were synthesized in deuterium‐ or tritium‐labeled form according to new protocols especially adapted to work without protection of the diphosphate moiety. When the labeled compounds MEcPP ( 7 ), 11 , and 12 were applied to chromoplast cultures, aldehyde 12 was not incorporated. This finding is in agreement with a mechanistic and structural model of the responsible enzyme family: a three‐dimensional model of the fragment L271–A375 of the enzyme GcpE of Streptomyces coelicolor including NADPH, the Fe 4 S 4 cluster, and MEcPP ( 7 ) as ligand has been developed based on homology modeling techniques. The model has been accepted by the Protein Data Bank (entry code 1OX2). Supported by this model, semiempirical PM3 calculations were performed to analyze the likely catalysis mechanism of the reductive ring opening of MEcPP ( 7 ), hydroxyl abstraction, and formation of HMBPP ( 8 ). The mechanism is characterized by a proton transfer (presumably from a conserved arginine 286) to the substrate, accompanied by a ring opening without high energy barriers, followed by the transfer of two electrons delivered from the Fe 4 S 4 cluster, and finally proton transfer from a carboxylic acid side chain to the hydroxyl group to be removed from the ligand as water. The proposed mechanism is in agreement with all known experimental findings and the arrangement of the ligand within the enzyme. Thus, a very likely mechanism for the second to last step of the DXP/MEP pathway in isoprenoid biosynthesis in plants is presented. A principally similar mechanism is also expected for the reductive dehydroxylation of HMBPP ( 8 ) to IPP ( 9 ) and DMAPP ( 10 ) in the last step.