scholarly journals Structural Analysis of Saccharomyces cerevisiae Dihydroorotase Reveals Molecular Insights into the Tetramerization Mechanism

Molecules ◽  
2021 ◽  
Vol 26 (23) ◽  
pp. 7249
Author(s):  
Hong-Hsiang Guan ◽  
Yen-Hua Huang ◽  
En-Shyh Lin ◽  
Chun-Jung Chen ◽  
Cheng-Yang Huang
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Structural Analysis ◽  
Sequence Analysis ◽  
Crystal Structures ◽  
Active Site ◽  
Crystalline State ◽  
Structural Similarity ◽  
Asymmetric Unit

Dihydroorotase (DHOase), a dimetalloenzyme containing a carbamylated lysine within the active site, is a member of the cyclic amidohydrolase family, which also includes allantoinase (ALLase), dihydropyrimidinase (DHPase), hydantoinase, and imidase. Unlike most known cyclic amidohydrolases, which are tetrameric, DHOase exists as a monomer or dimer. Here, we report and analyze two crystal structures of the eukaryotic Saccharomyces cerevisiae DHOase (ScDHOase) complexed with malate. The structures of different DHOases were also compared. An asymmetric unit of these crystals contained four crystallographically independent ScDHOase monomers. ScDHOase shares structural similarity with Escherichia coli DHOase (EcDHOase). Unlike EcDHOase, ScDHOase can form tetramers, both in the crystalline state and in solution. In addition, the subunit-interacting residues of ScDHOase for dimerization and tetramerization are significantly different from those of other DHOases. The tetramerization pattern of ScDHOase is also different from those of DHPase and ALLase. Based on sequence analysis and structural evidence, we identify two unique helices (α6 and α10) and a loop (loop 7) for tetramerization, and discuss why the residues for tetramerization in ScDHOase are not necessarily conserved among DHOases.

scholarly journals Crystal structures of two monomeric triosephosphate isomerase variants identifiedviaa directed-evolution protocol selecting forL-arabinose isomerase activity

2016 ◽  
Vol 72 (6) ◽  
pp. 490-499
Author(s):  
Mirja Krause ◽  
Tiila-Riikka Kiema ◽  
Peter Neubauer ◽  
Rik K. Wierenga
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Directed Evolution ◽  
Crystal Structures ◽  
Active Site ◽  
Triosephosphate Isomerase ◽  
Point Mutations ◽  
Van Der Waals Interactions ◽  
Side Chain ◽  
Arabinose Isomerase

The crystal structures are described of two variants of A-TIM: Ma18 (2.7 Å resolution) and Ma21 (1.55 Å resolution). A-TIM is a monomeric loop-deletion variant of triosephosphate isomerase (TIM) which has lost the TIM catalytic properties. Ma18 and Ma21 were identified after extensive directed-evolution selection experiments using anEscherichia coliL-arabinose isomerase knockout strain expressing a randomly mutated A-TIM gene. These variants facilitate better growth of theEscherichia coliselection strain in medium supplemented with 40 mML-arabinose. Ma18 and Ma21 differ from A-TIM by four and one point mutations, respectively. Ma18 and Ma21 are more stable proteins than A-TIM, as judged from CD melting experiments. Like A-TIM, both proteins are monomeric in solution. In the Ma18 crystal structure loop 6 is open and in the Ma21 crystal structure loop 6 is closed, being stabilized by a bound glycolate molecule. The crystal structures show only small differences in the active site compared with A-TIM. In the case of Ma21 it is observed that the point mutation (Q65L) contributes to small structural rearrangements near Asn11 of loop 1, which correlate with different ligand-binding properties such as a loss of citrate binding in the active site. The Ma21 structure also shows that its Leu65 side chain is involved in van der Waals interactions with neighbouring hydrophobic side-chain moieties, correlating with its increased stability. The experimental data suggest that the increased stability and solubility properties of Ma21 and Ma18 compared with A-TIM cause better growth of the selection strain when coexpressing Ma21 and Ma18 instead of A-TIM.

scholarly journals Crystal structures of the editing domain of Escherichia coli leucyl-tRNA synthetase and its complexes with Met and Ile reveal a lock-and-key mechanism for amino acid discrimination

2006 ◽  
Vol 394 (2) ◽  
pp. 399-407 ◽  
Author(s):  
Yunqing Liu ◽  
Jing Liao ◽  
Bin Zhu ◽  
En-Duo Wang ◽  
Jianping Ding
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Crystal Structures ◽  
Active Site ◽  
Binding Pocket ◽  
Trna Synthetase ◽  
Vital Role ◽  
Common Mechanism ◽  
Trna Synthetases ◽  
Editing Domain

aaRSs (aminoacyl-tRNA synthetases) are responsible for the covalent linking of amino acids to their cognate tRNAs via the aminoacylation reaction and play a vital role in maintaining the fidelity of protein synthesis. LeuRS (leucyl-tRNA synthetase) can link not only the cognate leucine but also the nearly cognate residues Ile and Met to tRNALeu. The editing domain of LeuRS deacylates the mischarged Ile–tRNALeu and Met–tRNALeu. We report here the crystal structures of ecLeuRS-ED (the editing domain of Escherichia coli LeuRS) in both the apo form and in complexes with Met and Ile at 2.0 Å, 2.4 Å, and 3.2 Å resolution respectively. The editing active site consists of a number of conserved amino acids, which are involved in the precise recognition and binding of the noncognate amino acids. The substrate-binding pocket has a rigid structure which has an optimal stereochemical fit for Ile and Met, but has steric hindrance for leucine. Based on our structural results and previously available biochemical data, we propose that ecLeuRS-ED uses a lock-and-key mechanism to recognize and discriminate between the amino acids. Structural comparison also reveals that all subclass Ia aaRSs share a conserved structure core consisting of the editing domain and conserved residues at the editing active site, suggesting that these enzymes may use a common mechanism for the editing function.

Crystallization and preliminary crystallographic analysis of major nitroreductase from Escherichia coli

1999 ◽  
Vol 55 (11) ◽  
pp. 1901-1902 ◽  
Author(s):  
Toshiro Kobori ◽  
Woo Cheol Lee ◽  
Takako Akagi ◽  
Hiroshi Sasaki ◽  
Shuhei Zenno ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Polyethylene Glycol ◽  
Structural Analysis ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
X Rays ◽  
Asymmetric Unit ◽  
Triclinic Space Group ◽  
Cell Dimensions ◽  
Polyethylene Glycol 6000

NADPH:nitrocompound oxidoreductase from Escherichia coli, NfsA, has been crystallized in the presence of FMN by the vapor-diffusion method using polyethylene glycol 6000 as a precipitant. The crystals belonged to the triclinic space group P1 with cell dimensions, a = 52.2, b = 52.7, c = 53.3 Å, \alpha = 75.1, β = 60.1, \gamma = 60.5°. The crystals are expected to contain two NfsA molecules per asymmetric unit. The crystals diffracted X-rays to at least 2.3 Å resolution and are appropriate for structural analysis at high resolution.

Sequence analysis of temperature-sensitive mutations in the Saccharomyces cerevisiae gene CDC28

1986 ◽  
Vol 6 (11) ◽  
pp. 4099-4103
Author(s):  
A T Lörincz ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Structural Analysis ◽  
Sequence Analysis ◽  
Structural Transition ◽  
Point Mutations ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Type Gene

Eleven independently isolated temperature-sensitive mutations in the cell division cycle gene CDC28 were mapped with respect to the DNA sequence of the wild-type gene and then sequenced to determine the precise nature of each mutation. The set yielded six different point mutations, each of which predicts a single amino acid substitution in the CDC28 product. The positions of the mutations did not correlate in any obvious way with observable biological characteristics of the mutant alleles. When the positions of substitutions were collated with a predicted secondary structural analysis of the CDC28 protein kinase, they were found to correlate strongly with probable regions of structural transition.

scholarly journals Sequence Analysis of the Gene Encoding Amylosucrase from Neisseria polysaccharea and Characterization of the Recombinant Enzyme

1999 ◽  
Vol 181 (2) ◽  
pp. 375-381 ◽  
Author(s):  
G. Potocki De Montalk ◽  
M. Remaud-Simeon ◽  
R. M. Willemot ◽  
V. Planchot ◽  
P. Monsan
Keyword(s):  
Escherichia Coli ◽  
Sequence Analysis ◽  
Active Site ◽  
Degree Of Polymerization ◽  
Gene Encoding ◽  
Amylolytic Enzymes ◽  
Strong Homology ◽  
Branched Chains ◽  
Sepharose 4B

ABSTRACT The Neisseria polysaccharea gene encoding amylosucrase was subcloned and expressed in Escherichia coli. Sequencing revealed that the deduced amino acid sequence differs significantly from that previously published. Comparison of the sequence with that of enzymes of the α-amylase family predicted a (β/α)8-barrel domain. Six of the eight highly conserved regions in amylolytic enzymes are present in amylosucrase. Among them, four constitute the active site in α-amylases. These sites were also conserved in the sequence of glucosyltransferases and dextransucrases. Nevertheless, the evolutionary tree does not show strong homology between them. The amylosucrase was purified by affinity chromatography between fusion protein glutathioneS-transferase–amylosucrase and glutathione-Sepharose 4B. The pure enzyme linearly elongated some branched chains of glycogen, to an average degree of polymerization of 75.

scholarly journals Sequence analysis of temperature-sensitive mutations in the Saccharomyces cerevisiae gene CDC28.

1986 ◽  
Vol 6 (11) ◽  
pp. 4099-4103 ◽  
Author(s):  
A T Lörincz ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Structural Analysis ◽  
Sequence Analysis ◽  
Structural Transition ◽  
Point Mutations ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Type Gene

Eleven independently isolated temperature-sensitive mutations in the cell division cycle gene CDC28 were mapped with respect to the DNA sequence of the wild-type gene and then sequenced to determine the precise nature of each mutation. The set yielded six different point mutations, each of which predicts a single amino acid substitution in the CDC28 product. The positions of the mutations did not correlate in any obvious way with observable biological characteristics of the mutant alleles. When the positions of substitutions were collated with a predicted secondary structural analysis of the CDC28 protein kinase, they were found to correlate strongly with probable regions of structural transition.

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