scholarly journals Crystal structures of the active site mutant (Arg-243 → Ala) in the T and R allosteric states of pig kidney fructose-1,6-bisphosphatase expressed in escherichia coli

1996 ◽  
Vol 5 (8) ◽  
pp. 1541-1553 ◽  
Author(s):  
Boguslaw Stec ◽  
Reimar Abraham ◽  
Eugene Giroux ◽  
Evan R. Kantrowitz
Keyword(s):  
Escherichia Coli ◽  
Crystal Structures ◽  
Active Site ◽  
Pig Kidney ◽  
Fructose 1,6 Bisphosphatase

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.

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 ribonuclease HI active site mutants from Escherichia coli.

1993 ◽  
Vol 268 (29) ◽  
pp. 22092-22099
Author(s):  
K Katayanagi ◽  
M Ishikawa ◽  
M Okumura ◽  
M Ariyoshi ◽  
S Kanaya ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Crystal Structures ◽  
Active Site ◽  
Active Site Mutants

scholarly journals Structures of theMycobacterium tuberculosisGlpX protein (class II fructose-1,6-bisphosphatase): implications for the active oligomeric state, catalytic mechanism and citrate inhibition

2018 ◽  
Vol 74 (4) ◽  
pp. 321-331
Author(s):  
Nina M. Wolf ◽  
Hiten J. Gutka ◽  
Farahnaz Movahedzadeh ◽  
Celerino Abad-Zapatero
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Adenosine Monophosphate ◽  
Class Ii ◽  
Inhibitory Effect ◽  
Amino Acid Residues ◽  
Oligomeric State ◽  
Fructose 1,6 Bisphosphatase ◽  
Lead Inhibitors

The crystal structures of native class II fructose-1,6-bisphosphatase (FBPaseII) fromMycobacterium tuberculosisat 2.6 Å resolution and two active-site protein variants are presented. The variants were complexed with the reaction product fructose 6-phosphate (F6P). The Thr84Ala mutant is inactive, while the Thr84Ser mutant has a lower catalytic activity. The structures reveal the presence of a 222 tetramer, similar to those described for fructose-1,6/sedoheptulose-1,7-bisphosphatase fromSynechocystis(strain 6803) as well as the equivalent enzyme fromThermosynechococcus elongatus. This homotetramer corresponds to a homologous oligomer that is present but not described in the crystal structure of FBPaseII fromEscherichia coliand is probably conserved in all FBPaseIIs. The constellation of amino-acid residues in the active site of FBPaseII fromM. tuberculosis(MtFBPaseII) is conserved and is analogous to that described previously for theE. colienzyme. Moreover, the structure of the active site of the partially active (Thr84Ser) variant and the analysis of the kinetics are consistent with the previously proposed catalytic mechanism. The presence of metabolites in the crystallization medium (for example citrate and malonate) and in the corresponding crystal structures ofMtFBPaseII, combined with their observed inhibitory effect, could suggest the existence of an uncharacterized inhibition of this class of enzymes besides the allosteric inhibition by adenosine monophosphate observed for theSynechocystisenzyme. The structural and functional insights derived from the structure ofMtFBPaseII will provide critical information for the design of lead inhibitors, which will be used to validate this target for future chemical intervention.

scholarly journals Major reorientation of tRNA substrates defines specificity of dihydrouridine synthases

2015 ◽  
Vol 112 (19) ◽  
pp. 6033-6037 ◽  
Author(s):  
Robert T. Byrne ◽  
Huw T. Jenkins ◽  
Daniel T. Peters ◽  
Fiona Whelan ◽  
James Stowell ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Substrate Specificity ◽  
Crystal Structures ◽  
Active Site ◽  
Protein Fold ◽  
Catalytic Center ◽  
Trna Recognition ◽  
Trna Binding ◽  
Positively Charged

The reduction of specific uridines to dihydrouridine is one of the most common modifications in tRNA. Increased levels of the dihydrouridine modification are associated with cancer. Dihydrouridine synthases (Dus) from different subfamilies selectively reduce distinct uridines, located at spatially unique positions of folded tRNA, into dihydrouridine. Because the catalytic center of all Dus enzymes is conserved, it is unclear how the same protein fold can be reprogrammed to ensure that nucleotides exposed at spatially distinct faces of tRNA can be accommodated in the same active site. We show that the Escherichia coli DusC is specific toward U16 of tRNA. Unexpectedly, crystal structures of DusC complexes with tRNAPhe and tRNATrp show that Dus subfamilies that selectively modify U16 or U20 in tRNA adopt identical folds but bind their respective tRNA substrates in an almost reverse orientation that differs by a 160° rotation. The tRNA docking orientation appears to be guided by subfamily-specific clusters of amino acids (“binding signatures”) together with differences in the shape of the positively charged tRNA-binding surfaces. tRNA orientations are further constrained by positional differences between the C-terminal “recognition” domains. The exquisite substrate specificity of Dus enzymes is therefore controlled by a relatively simple mechanism involving major reorientation of the whole tRNA molecule. Such reprogramming of the enzymatic specificity appears to be a unique evolutionary solution for altering tRNA recognition by the same protein fold.

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