scholarly journals Structures of trehalose synthase fromDeinococcus radioduransreveal that a closed conformation is involved in catalysis of the intramolecular isomerization

2014 ◽  
Vol 70 (12) ◽  
pp. 3144-3154 ◽  
Author(s):  
Yung-Lin Wang ◽  
Sih-Yao Chow ◽  
Yi-Ting Lin ◽  
Yu-Chiao Hsieh ◽  
Guan-Chiun Lee ◽  
...  
Keyword(s):  
Active Site ◽  
Deinococcus Radiodurans ◽  
Mutational Analysis ◽  
Side Reaction ◽  
Trehalose Synthase ◽  
Isomerase Activity ◽  
Closed Conformation ◽  
Dimeric Interface ◽  
Small Pore ◽  
Simple Conversion

Trehalose synthase catalyzes the simple conversion of the inexpensive maltose into trehalose with a side reaction of hydrolysis. Here, the crystal structures of the wild type and the N253A mutant ofDeinococcus radioduranstrehalose synthase (DrTS) in complex with the inhibitor Tris are reported. DrTS consists of a catalytic (β/α)8barrel, subdomain B, a C-terminal β domain and two TS-unique subdomains (S7 and S8). The C-terminal domain and S8 contribute the majority of the dimeric interface. DrTS shares high structural homology with sucrose hydrolase, amylosucrase and sucrose isomerase in complex with sucrose, in particular a virtually identical active-site architecture and a similar substrate-induced rotation of subdomain B. The inhibitor Tris was bound and mimics a sugar at the −1 subsite. A maltose was modelled into the active site, and subsequent mutational analysis suggested that Tyr213, Glu320 and Glu324 are essential within the +1 subsite for the TS activity. In addition, the interaction networks between subdomains B and S7 seal the active-site entrance. Disruption of such networks through the replacement of Arg148 and Asn253 with alanine resulted in a decrease in isomerase activity by 8–9-fold and an increased hydrolase activity by 1.5–1.8-fold. The N253A structure showed a small pore created for water entry. Therefore, our DrTS-Tris may represent a substrate-induced closed conformation that will facilitate intramolecular isomerization and minimize disaccharide hydrolysis.

scholarly journals The N253F mutant structure of trehalose synthase fromDeinococcus radioduransreveals an open active-site topology

2017 ◽  
Vol 73 (11) ◽  
pp. 588-594
Author(s):  
Sih-Yao Chow ◽  
Yung-Lin Wang ◽  
Yu-Chiao Hsieh ◽  
Guan-Chiun Lee ◽  
Shwu-Huey Liaw
Keyword(s):  
Active Site ◽  
Deinococcus Radiodurans ◽  
Glycoside Hydrolase Family ◽  
Trehalose Synthase ◽  
Site Analysis ◽  
Mechanistic Analysis ◽  
Opening And Closing ◽  
Rate Limiting ◽  
Hydrolase Family ◽  
Domain Rotation

Trehalose synthase (TS) catalyzes the reversible conversion of maltose to trehalose and belongs to glycoside hydrolase family 13 (GH13). Previous mechanistic analysis suggested a rate-limiting protein conformational change, which is probably the opening and closing of the active site. Consistently, crystal structures ofDeinococcus radioduransTS (DrTS) in complex with the inhibitor Tris displayed an enclosed active site for catalysis of the intramoleular isomerization. In this study, the apo structure of the DrTS N253F mutant displays a new open conformation with an empty active site. Analysis of these structures suggests that substrate binding induces a domain rotation to close the active site. Such a substrate-induced domain rotation has also been observed in some other GH13 enzymes.

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

Active site separation of photocatalytic steam reforming of methane using Gas‐Phase Photoelectrochemical system

2021 ◽  
Author(s):  
Tomoki Kujirai ◽  
Akira Yamaguchi ◽  
Takeshi Fujita ◽  
Hideki Abe ◽  
Masahiro Miyauchi
Keyword(s):  
Carbon Dioxide ◽  
Gas Phase ◽  
High Temperatures ◽  
Steam Reforming ◽  
Active Site ◽  
Side Reaction ◽  
System P ◽  
Steam Reforming Of Methane

Steam reforming of methane (SRM) requires high temperatures to be promoted, and the production of carbon dioxide from the side reaction has also become a problem. In this study, we...

scholarly journals Structural and Biochemical Characterization of Thioredoxin-2 from Deinococcus radiodurans

Antioxidants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1843
Author(s):  
Min-Kyu Kim ◽  
Lei Zhao ◽  
Soyoung Jeong ◽  
Jing Zhang ◽  
Jong-Hyun Jung ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Active Site ◽  
Biochemical Characterization ◽  
Deinococcus Radiodurans ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Antioxidant Systems ◽  
Zinc Finger Domain ◽  
Cxxc Motif ◽  
Reduction Activity ◽  
Trx Fold

Thioredoxin (Trx), a ubiquitous protein showing disulfide reductase activity, plays critical roles in cellular redox control and oxidative stress response. Trx is a member of the Trx system, comprising Trx, Trx reductase (TrxR), and a cognate reductant (generally reduced nicotinamide adenine dinucleotide phosphate, NADPH). Bacterial Trx1 contains only the Trx-fold domain, in which the active site CXXC motif that is critical for the disulfide reduction activity is located. Bacterial Trx2 contains an N-terminal extension, which forms a zinc-finger domain, including two additional CXXC motifs. The multi-stress resistant bacterium Deinococcus radiodurans encodes both Trx1 (DrTrx1) and Trx2 (DrTrx2), which act as members of the enzymatic antioxidant systems. In this study, we constructed Δdrtrx1 and Δdrtrx2 mutants and examined their survival rates under H2O2 treated conditions. Both drtrx1 and drtrx2 genes were induced following H2O2 treatment, and the Δdrtrx1 and Δdrtrx2 mutants showed a decrease in resistance toward H2O2, compared to the wild-type. Native DrTrx1 and DrTrx2 clearly displayed insulin and DTNB reduction activity, whereas mutant DrTrx1 and DrTrx2, which harbors the substitution of conserved cysteine to serine in its active site CXXC motif, showed almost no reduction activity. Mutations in the zinc binding cysteines did not fully eliminate the reduction activities of DrTrx2. Furthermore, we solved the crystal structure of full-length DrTrx2 at 1.96 Å resolution. The N-terminal zinc-finger domain of Trx2 is thought to be involved in Trx-target interaction and, from our DrTrx2 structure, the orientation of the zinc-finger domain of DrTrx2 and its interdomain interaction, between the Trx-fold domain and the zinc-finger domain, is clearly distinguished from those of the other Trx2 structures.

scholarly journals Not Cleaving the His-tag of Thal Results in More Tightly Packed and Better-Diffracting Crystals

Crystals ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1135
Author(s):  
Ann-Christin Moritzer ◽  
Tina Prior ◽  
Hartmut H. Niemann
Keyword(s):  
Active Site ◽  
Flavin Adenine Dinucleotide ◽  
Substrate Binding ◽  
Environmentally Friendly ◽  
Space Group ◽  
Cofactor Binding ◽  
Closed Conformation ◽  
His Tag ◽  
Halide Salts ◽  
Binding Loop

Flavin-dependent halogenases chlorinate or brominate their substrates in an environmentally friendly manner, only requiring the cofactor reduced flavin adenine dinucleotide (FADH2), oxygen, and halide salts. The tryptophan 6-halogenase Thal exhibits two flexible loops, which become ordered (substrate-binding loop) or adopt a closed conformation (FAD loop) upon substrate or cofactor binding. Here, we describe the structure of NHis-Thal-RebH5 containing an N-terminal His-tag from pET28a, which crystallized in a different space group (P21) and, surprisingly, diffracted to a higher resolution of 1.63 Å than previously deposited Thal structures (P64; ~2.2 Å) with cleaved His-tag. Interestingly, the binding of glycine in the active site can induce an ordered conformation of the substrate-binding loop.

scholarly journals A Mutational Analysis of the Active Site Loop Residues incis-3-Chloroacrylic Acid Dehalogenase

Biochemistry ◽  
2013 ◽  
Vol 52 (24) ◽  
pp. 4204-4216 ◽  
Author(s):  
Gottfried K. Schroeder ◽  
Jamison P. Huddleston ◽  
William H. Johnson ◽  
Christian P. Whitman
Keyword(s):  
Active Site ◽  
Mutational Analysis ◽  
Chloroacrylic Acid Dehalogenase
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