scholarly journals Crystal Structure of Tetrameric Homoisocitrate Dehydrogenase from an Extreme Thermophile, Thermus thermophilus: Involvement of Hydrophobic Dimer-Dimer Interaction in Extremely High Thermotolerance

2005 ◽  
Vol 187 (19) ◽  
pp. 6779-6788 ◽  
Author(s):  
Junichi Miyazaki ◽  
Kuniko Asada ◽  
Shinya Fushinobu ◽  
Tomohisa Kuzuyama ◽  
Makoto Nishiyama
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Thermal Inactivation ◽  
Hydrophobic Interactions ◽  
Substrate Binding ◽  
Thermus Thermophilus ◽  
Lysine Biosynthesis ◽  
Side Chain ◽  
Substrate Binding Site ◽  
Tetramer Formation

ABSTRACT The crystal structure of homoisocitrate dehydrogenase involved in lysine biosynthesis from Thermus thermophilus (TtHICDH) was determined at 1.85-Å resolution. Arg85, which was shown to be a determinant for substrate specificity in our previous study, is positioned close to the putative substrate binding site and interacts with Glu122. Glu122 is highly conserved in the equivalent position in the primary sequence of ICDH and archaeal 3-isopropylmalate dehydrogenase (IPMDH) but interacts with main- and side-chain atoms in the same domain in those paralogs. In addition, a conserved Tyr residue (Tyr125 in TtHICDH) which extends its side chain toward a substrate and thus has a catalytic function in the related β-decarboxylating dehydrogenases, is flipped out of the substrate-binding site. These results suggest the possibility that the conformation of the region containing Glu122-Tyr125 is changed upon substrate binding in TtHICDH. The crystal structure of TtHICDH also reveals that the arm region is involved in tetramer formation via hydrophobic interactions and might be responsible for the high thermotolerance. Mutation of Val135, located in the dimer-dimer interface and involved in the hydrophobic interaction, to Met alters the enzyme to a dimer (probably due to steric perturbation) and markedly decreases the thermal inactivation temperature. Both the crystal structure and the mutation analysis indicate that tetramer formation is involved in the extremely high thermotolerance of TtHICDH.

Enhancement of the latent 3-isopropylmalate dehydrogenase activity of promiscuous homoisocitrate dehydrogenase by directed evolution

2010 ◽  
Vol 431 (3) ◽  
pp. 401-412 ◽  
Author(s):  
Yumewo Suzuki ◽  
Kuniko Asada ◽  
Junichi Miyazaki ◽  
Takeo Tomita ◽  
Tomohisa Kuzuyama ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acid ◽  
Binding Site ◽  
Directed Evolution ◽  
Substrate Binding ◽  
Dna Shuffling ◽  
Turnover Number ◽  
Substrate Binding Site ◽  
Km Value ◽  
Isopropylmalate Dehydrogenase

HICDH (homoisocitrate dehydrogenase), which is involved in lysine biosynthesis through α-aminoadipate, is a paralogue of IPMDH [3-IPM (3-isopropylmalate) dehydrogenase], which is involved in leucine biosynthesis. TtHICDH (Thermus thermophilus HICDH) can recognize isocitrate, as well as homoisocitrate, as the substrate, and also shows IPMDH activity, although at a considerably decreased rate. In the present study, the promiscuous TtHICDH was evolved into an enzyme showing distinct IPMDH activity by directed evolution using a DNA-shuffling technique. Through five repeats of DNA shuffling/screening, variants that allowed Escherichia coli C600 (leuB−) to grow on a minimal medium in 2 days were obtained. One of the variants LR5–1, with eight amino acid replacements, was found to possess a 65-fold increased kcat/Km value for 3-IPM, compared with TtHICDH. Introduction of a single back-replacement H15Y change caused a further increase in the kcat/Km value and a partial recovery of the decreased thermotolerance of LR5–1. Site-directed mutagenesis revealed that most of the amino acid replacements found in LR5–1 effectively increased IPMDH activity; replacements around the substrate-binding site contributed to the improved recognition for 3-IPM, and other replacements at sites away from the substrate-binding site enhanced the turnover number for the IPMDH reaction. The crystal structure of LR5–1 was determined at 2.4 Å resolution and revealed that helix α4 was displaced in a manner suitable for recognition of the hydrophobic γ-moiety of 3-IPM. On the basis of the crystal structure, possible reasons for enhancement of the turnover number are discussed.

The 5-ketofructose reductase of Gluconobacter sp. strain CHM43 is a novel class in the shikimate dehydrogenase family

2021 ◽  
Author(s):  
Thuy Minh Nguyen ◽  
Masaru Goto ◽  
Shohei Noda ◽  
Minenosuke Matsutani ◽  
Yuki Hodoya ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acid ◽  
Binding Site ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
High Yield ◽  
Strong Increase ◽  
Amino Acid Residues ◽  
Shikimate Dehydrogenase ◽  
Substrate Binding Site

Gluconobacter sp. CHM43 oxidizes mannitol to fructose and then does fructose to 5-keto-D-fructose (5KF) in the periplasmic space. Since NADPH-dependent 5KF reductase was found in the soluble fraction of Gluconobacter spp., 5KF might be transported into the cytoplasm and metabolized. Here we identified the GLF_2050 gene as the kfr gene encoding 5KF reductase (KFR). A mutant strain devoid of the kfr gene showed lower KFR activity and no 5KF consumption. The crystal structure revealed that KFR is similar to NADP + -dependent shikimate dehydrogenase (SDH), which catalyzes the reversible NADP + -dependent oxidation of shikimate to 3-dehydroshikimate. We found that several amino acid residues in the putative substrate-binding site of KFR were different from those of SDH. Phylogenetic analyses revealed that only a subclass in the SDH family containing KFR conserved such a unique substrate-binding site. We constructed KFR derivatives with amino acid substitutions, including replacement of Asn21 in the substrate-binding site with Ser that is found in SDH. The KFR-N21S derivative showed a strong increase in the K M value for 5KF, but a higher shikimate oxidation activity than wild-type KFR, suggesting that Asn21 is important for 5KF binding. In addition, the conserved catalytic dyad Lys72 and Asp108 were individually substituted for Asn. The K72N and D108N derivatives showed only negligible activities without a dramatic change in the K M value for 5KF, suggesting a similar catalytic mechanism to that of SDH. Taken together, we suggest that KFR is a new member of the SDH family. Importance A limited number of species of acetic acid bacteria, such as Gluconobacter sp. strain CHM43, produce 5-ketofructose at a high yield, a potential low calorie sweetener. Here we show that an NADPH-dependent 5-ketofructose reductase (KFR) is involved in 5-ketofructose degradation and we characterize this enzyme with respect to its structure, phylogeny, and function. The crystal structure of KFR was similar to that of shikimate dehydrogenase, which is functionally crucial in the shikimate pathway in bacteria and plants. Phylogenetic analysis suggested that KFR is positioned in a small sub-group of the shikimate dehydrogenase family. Catalytically important amino acid residues were also conserved and their relevance was experimentally validated. Thus, we propose KFR as a new member of shikimate dehydrogenase family.

scholarly journals Cryo-EM structures of Escherichia coli cytochrome bo3 reveal bound phospholipids and ubiquinone-8 in a dynamic substrate binding site

2021 ◽  
Vol 118 (34) ◽  
pp. e2106750118 ◽  
Author(s):  
Jiao Li ◽  
Long Han ◽  
Francesca Vallese ◽  
Ziqiao Ding ◽  
Sylvia K. Choi ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Binding Site ◽  
Substrate Binding ◽  
Transmembrane Helix ◽  
Proton Pumping ◽  
Side Chain ◽  
Substrate Binding Site ◽  
Cytochrome Bo3 ◽  
Redox Active ◽  
Quinol Oxidases

Two independent structures of the proton-pumping, respiratory cytochrome bo3 ubiquinol oxidase (cyt bo3) have been determined by cryogenic electron microscopy (cryo-EM) in styrene–maleic acid (SMA) copolymer nanodiscs and in membrane scaffold protein (MSP) nanodiscs to 2.55- and 2.19-Å resolution, respectively. The structures include the metal redox centers (heme b, heme o3, and CuB), the redox-active cross-linked histidine–tyrosine cofactor, and the internal water molecules in the proton-conducting D channel. Each structure also contains one equivalent of ubiquinone-8 (UQ8) in the substrate binding site as well as several phospholipid molecules. The isoprene side chain of UQ8 is clamped within a hydrophobic groove in subunit I by transmembrane helix TM0, which is only present in quinol oxidases and not in the closely related cytochrome c oxidases. Both structures show carbonyl O1 of the UQ8 headgroup hydrogen bonded to D75I and R71I. In both structures, residue H98I occupies two conformations. In conformation 1, H98I forms a hydrogen bond with carbonyl O4 of the UQ8 headgroup, but in conformation 2, the imidazole side chain of H98I has flipped to form a hydrogen bond with E14I at the N-terminal end of TM0. We propose that H98I dynamics facilitate proton transfer from ubiquinol to the periplasmic aqueous phase during oxidation of the substrate. Computational studies show that TM0 creates a channel, allowing access of water to the ubiquinol headgroup and to H98I.

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