Crystal structure of dimeric Synechococcus spermidine synthase with bound polyamine substrate and product

2019 ◽  
Vol 476 (6) ◽  
pp. 1009-1020 ◽  
Author(s):  
Gabriela Guédez ◽  
Apiradee Pothipongsa ◽  
Saija Sirén ◽  
Arto Liljeblad ◽  
Saowarath Jantaro ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Crystal Structures ◽  
Catalytic Reaction ◽  
Active Site ◽  
Enzymatic Reaction ◽  
Spermidine Synthase ◽  
Structural Element ◽  
Expression Host ◽  
Gate Keeping

AbstractSpermidine is a ubiquitous polyamine synthesized by spermidine synthase (SPDS) from the substrates, putrescine and decarboxylated S-adenosylmethionine (dcAdoMet). SPDS is generally active as homodimer, but higher oligomerization states have been reported in SPDS from thermophiles, which are less specific to putrescine as the aminoacceptor substrate. Several crystal structures of SPDS have been solved with and without bound substrates and/or products as well as inhibitors. Here, we determined the crystal structure of SPDS from the cyanobacterium Synechococcus (SySPDS) that is a homodimer, which we also observed in solution. Unlike crystal structures reported for bacterial and eukaryotic SPDS with bound ligands, SySPDS structure has not only bound putrescine substrate taken from the expression host, but also spermidine product most probably as a result of an enzymatic reaction. Hence, to the best of our knowledge, this is the first structure reported with both amino ligands in the same structure. Interestingly, the gate-keeping loop is disordered in the putrescine-bound monomer while it is stabilized in the spermidine-bound monomer of the SySPDS dimer. This confirms the gate-keeping loop as the key structural element that prepares the active site upon binding of dcAdoMet for the catalytic reaction of the amine donor and putrescine.

scholarly journals How cyanophage S-2L rejects adenine and incorporates 2-aminoadenine to saturate hydrogen bonding in its DNA

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dariusz Czernecki ◽  
Pierre Legrand ◽  
Mustafa Tekpinar ◽  
Sandrine Rosario ◽  
Pierre-Alexandre Kaminski ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bonding ◽  
Crystal Structures ◽  
Active Site ◽  
Metal Ion ◽  
Restriction Endonucleases ◽  
Structural Element

AbstractBacteriophages have long been known to use modified bases in their DNA to prevent cleavage by the host’s restriction endonucleases. Among them, cyanophage S-2L is unique because its genome has all its adenines (A) systematically replaced by 2-aminoadenines (Z). Here, we identify a member of the PrimPol family as the sole possible polymerase of S-2L and we find it can incorporate both A and Z in front of a T. Its crystal structure at 1.5 Å resolution confirms that there is no structural element in the active site that could lead to the rejection of A in front of T. To resolve this contradiction, we show that a nearby gene is a triphosphohydolase specific of dATP (DatZ), that leaves intact all other dNTPs, including dZTP. This explains the absence of A in S-2L genome. Crystal structures of DatZ with various ligands, including one at sub-angstrom resolution, allow to describe its mechanism as a typical two-metal-ion mechanism and to set the stage for its engineering.

The crystal structure of Proteus vulgaris tryptophan indole-lyase complexed with oxindolyl-L-alanine: implications for the reaction mechanism

2018 ◽  
Vol 74 (8) ◽  
pp. 748-759
Author(s):  
Robert S. Phillips ◽  
Adriaan A. Buisman ◽  
Sarah Choi ◽  
Anusha Hussaini ◽  
Zachary A. Wood
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bond ◽  
Aromatic Ring ◽  
Active Site ◽  
Enzymatic Reaction ◽  
Proteus Vulgaris ◽  
Bacterial Enzyme ◽  
Small Domain ◽  
Out Of Plane ◽  
Reversible Formation

Tryptophan indole-lyase (TIL) is a bacterial enzyme which catalyzes the reversible formation of indole and ammonium pyruvate from L-tryptophan. Oxindolyl-L-alanine (OIA) is an inhibitor of TIL, with a K i value of about 5 µM. The crystal structure of the complex of Proteus vulgaris TIL with OIA has now been determined at 2.1 Å resolution. The ligand forms a closed quinonoid complex with the pyridoxal 5′-phosphate (PLP) cofactor. The small domain rotates about 10° to close the active site, bringing His458 into position to donate a hydrogen bond to Asp133, which also accepts a hydrogen bond from the heterocyclic NH of the inhibitor. This brings Phe37 and Phe459 into van der Waals contact with the aromatic ring of OIA. Mutation of the homologous Phe464 in Escherichia coli TIL to Ala results in a 500-fold decrease in k cat/K m for L-tryptophan, with less effect on the reaction of other nonphysiological β-elimination substrates. Stopped-flow kinetic experiments of F464A TIL show that the mutation has no effect on the formation of quinonoid intermediates. An aminoacrylate intermediate is observed in the reaction of F464A TIL with S-ethyl-L-cysteine and benzimidazole. A model of the L-tryptophan quinonoid complex with PLP in the active site of P. vulgaris TIL shows that there would be a severe clash of Phe459 (∼1.5 Å apart) and Phe37 (∼2 Å apart) with the benzene ring of the substrate. It is proposed that this creates distortion of the substrate aromatic ring out of plane and moves the substrate upwards on the reaction coordinate towards the transition state, thus reducing the activation energy and accelerating the enzymatic reaction.

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.

SELECTION OF BEST CRYSTAL STRUCTURE FOR INITIATING DOCKING-BASED VIRTUAL SCREENING STUDIES OF CDK2 INHIBITORS: A CROSS-DOCKING AND DUD SET VALIDATION APPROACH

INDIAN DRUGS ◽  
2019 ◽  
Vol 56 (06) ◽  
pp. 77-85
Author(s):  
A. Joshi ◽  
◽  
H Bhojwani ◽  
U Joshi
Keyword(s):  
Crystal Structure ◽  
Virtual Screening ◽  
Crystal Structures ◽  
Active Site ◽  
Docking Studies ◽  
Cross Docking ◽  
Screening Protocol ◽  
Initial Crystal ◽  
Docking Accuracy ◽  
Selection Of

A total of 95 crystal structures of CDK2 were selected after considering criteria such as resolution and absence of missing residues in the active site; and subjected to cross-docking. 14 out of 95 crystal structures exhibited docking accuracy for greater than 70% of ligands at RMSD cut off 2Å in the cross- docking studies. These 14 crystal structures were selected for the second part of the study, which included validation using DUD sets and enrichment calculations. 8 out of 14 crystal structures possessed the enrichment factor of >10 at 1% of the ranked database. ROC-AUC, AUAC, RIE, and BEDROC were calculated for these 8 crystal structures. 2WXV produced maximum BEDROC (0.768, at α=8) and RIE (11.22). 2WXV as a single initial crystal structure in the virtual screening protocol is likely to produce more accurate results than any other single crystal structure.

Crystal structure of Helicobacter pylori spermidine synthase: A Rossmann-like fold with a distinct active site

2007 ◽  
Vol 67 (3) ◽  
pp. 743-754 ◽  
Author(s):  
Po Kai Lu ◽  
Jia-Yin Tsai ◽  
Hsiang Yi Chien ◽  
Haimei Huang ◽  
Chen-Hsi Chu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Helicobacter Pylori ◽  
Active Site ◽  
Spermidine Synthase

scholarly journals Roles of the hydroxy group of tyrosine in crystal structures of Sulfurisphaera tokodaii O 6-methylguanine-DNA methyltransferase

2021 ◽  
Vol 77 (12) ◽  
Author(s):  
Makiko Kikuchi ◽  
Takahiro Yamauchi ◽  
Yasuhito Iizuka ◽  
Masaru Tsunoda
Keyword(s):  
Crystal Structure ◽  
Crystal Structures ◽  
Hydroxy Group ◽  
Active Site ◽  
Dna Methyltransferase ◽  
Structural Changes ◽  
Molecular Mechanisms ◽  
Alkylating Agents ◽  
Substrate Analogs ◽  
Methylguanine Dna Methyltransferase

O 6-Methylguanine-DNA methyltransferase (MGMT) removes cytotoxic O 6-alkyl adducts on the guanine base and protects the cell from genomic damage induced by alkylating agents. Although there are reports of computational studies on the activity of the enzyme with mutations at tyrosine residues, no studies concerning the crystal structure of its mutants have been found. In this study, the function of Tyr91 was investigated in detail by comparing the crystal structures of mutants and their complexes with substrate analogs. In this study, tyrosine, a conserved amino acid near the active-site loop in the C-terminal domain of Sulfurisphaera tokodaii MGMT (StoMGMT), was mutated to phenylalanine to produce a Y91F mutant, and the cysteine which is responsible for receiving the methyl group in the active site was mutated to a serine to produce a C120S mutant. A Y91F/C120S double-mutant StoMGMT was also created. The function of tyrosine is discussed based on the crystal structure of Y91F mutant StoMGMT. The crystal structures of StoMGMT were determined at resolutions of 1.13–2.60 Å. They showed no structural changes except in the mutated part. No electron density for deoxyguanosine or methyl groups was observed in the structure of Y91F mutant crystals immersed in O 6-methyl-2′-deoxyguanosine, nor was the group oxidized in wild-type StoMGMT. Therefore, the hydroxy group of Tyr91 may prevent the oxidant from entering the active site. This suggests that tyrosine, which is highly conserved at the N-terminus of the helix–turn–helix motif across species, protects the active site of MGMTs, which are deactivated after repairing only one alkyl adduct. Overall, the results may provide a basis for understanding the molecular mechanisms by which high levels of conserved amino acids play a role in ensuring the integrity of suicide enzymes, in addition to promoting their activity.

scholarly journals Structural analysis of the SARS-CoV-2 methyltransferase complex involved in coronaviral RNA cap creation

2020 ◽  
Author(s):  
Petra Krafcikova ◽  
Jan Silhan ◽  
Radim Nencka ◽  
Evzen Boura
Keyword(s):  
Crystal Structure ◽  
Structural Analysis ◽  
Catalytic Reaction ◽  
Active Site ◽  
Rna Viruses ◽  
Rna Stability ◽  
Structural Data ◽  
Nonstructural Proteins ◽  
Structural Comparison ◽  
Structure Based Drug Design

AbstractCOVID-19 pandemic is caused by the SARS-CoV-2 virus that has several enzymes that could be targeted by antivirals including a 2’-O RNA methyltransferase (MTase) that is involved in the viral RNA cap formation; an essential process for RNA stability. This MTase is composed of two nonstructural proteins, the nsp16 catalytic subunit and the activating nsp10 protein. We have solved the crystal structure of the nsp10-nsp16 complex bound to the pan-MTase inhibitor sinefungin in the active site. Based on the structural data we built a model of the MTase in complex with RNA that illustrates the catalytic reaction. A structural comparison to the Zika MTase revealed low conservation of the catalytic site between these two RNA viruses suggesting preparation of inhibitors targeting both these viruses will be very difficult. Together, our data will provide the information needed for structure-based drug design.

scholarly journals Crystal structures of the selenoprotein glutathione peroxidase 4 in its apo form and in complex with the covalently bound inhibitor ML162

2021 ◽  
Vol 77 (2) ◽  
pp. 237-248
Author(s):  
Dieter Moosmayer ◽  
André Hilpmann ◽  
Jutta Hoffmann ◽  
Lennart Schnirch ◽  
Katja Zimmermann ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mass Spectrometry ◽  
Glutathione Peroxidase ◽  
Crystal Structures ◽  
Active Site ◽  
Cancer Drug ◽  
Efficient Production ◽  
Wild Type ◽  
Preparative Scale ◽  
Covalent Inhibitors

Wild-type human glutathione peroxidase 4 (GPX4) was co-expressed with SBP2 (selenocysteine insertion sequence-binding protein 2) in human HEK cells to achieve efficient production of this selenocysteine-containing enzyme on a preparative scale for structural biology. The protein was purified and crystallized, and the crystal structure of the wild-type form of GPX4 was determined at 1.0 Å resolution. The overall fold and the active site are conserved compared with previously determined crystal structures of mutated forms of GPX4. A mass-spectrometry-based approach was developed to monitor the reaction of the active-site selenocysteine Sec46 with covalent inhibitors. This, together with the introduction of a surface mutant (Cys66Ser), enabled the crystal structure determination of GPX4 in complex with the covalent inhibitor ML162 [(S)-enantiomer]. The mass-spectrometry-based approach described here opens the path to further co-complex crystal structures of this potential cancer drug target in complex with covalent inhibitors.

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