Crystal structure and molecular mechanism of phosphotransbutyrylase from Clostridium acetobutylicum

Crystalline N-(para-chlorophenyl) phthalanilic acid undergoes a topochemical cyclization reaction with the elimination of water yielding crystalline N-(para-chlorophenyl) phthalimide. The crystal structure of the imide product has been determined and compared to the previously determined structure of the reactant. The reaction from this particular crystalline phase of N-(para-chlorophenyl) phthalanilic acid is enhanced by the presence of a high energy molecular conformer of the acid, little molecular motion necessary for reaction, and only a minor rearrangement required to pack into the imide structure after the reaction. A detailed molecular mechanism for this reaction is proposed involving some bond rotation, but little movement of the molecule as a whole.

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Structural Analysis of an L-Cysteine Desulfurase from an Ssp DNA Phosphorothioation System

mBio ◽

10.1128/mbio.00488-20 ◽

2020 ◽

Vol 11 (2) ◽

Author(s):

Liqiong Liu ◽

Susu Jiang ◽

Mai Xing ◽

Chao Chen ◽

Chongde Lai ◽

...

Keyword(s):

Crystal Structure ◽

Conformational Change ◽

Molecular Mechanism ◽

Sulfur Atom ◽

Nucleophilic Attack ◽

Nonbridging Oxygen ◽

Long Distance ◽

Cysteine Desulfurase ◽

Modification System ◽

Pt Modification

ABSTRACT DNA phosphorothioate (PT) modification, in which the nonbridging oxygen in the sugar-phosphate backbone is substituted by sulfur, is catalyzed by DndABCDE or SspABCD in a double-stranded or single-stranded manner, respectively. In Dnd and Ssp systems, mobilization of sulfur in PT formation starts with the activation of the sulfur atom of cysteine catalyzed by the DndA and SspA cysteine desulfurases, respectively. Despite playing the same biochemical role, SspA cannot be functionally replaced by DndA, indicating its unique physiological properties. In this study, we solved the crystal structure of Vibrio cyclitrophicus SspA in complex with its natural substrate, cysteine, and cofactor, pyridoxal phosphate (PLP), at a resolution of 1.80 Å. Our solved structure revealed the molecular mechanism that SspA employs to recognize its cysteine substrate and PLP cofactor, suggesting a common binding mode shared by cysteine desulfurases. In addition, although the distance between the catalytic Cys314 and the substrate cysteine is 8.9 Å, which is too far for direct interaction, our structural modeling and biochemical analysis revealed a conformational change in the active site region toward the cysteine substrate to move them close to each other to facilitate the nucleophilic attack. Finally, the pulldown analysis showed that SspA could form a complex with SspD, an ATP pyrophosphatase, suggesting that SspD might potentially accept the activated sulfur atom directly from SspA, providing further insights into the biochemical pathway of Ssp-mediated PT modification. IMPORTANCE Apart from its roles in Fe-S cluster assembly, tRNA thiolation, and sulfur-containing cofactor biosynthesis, cysteine desulfurase serves as a sulfur donor in the DNA PT modification, in which a sulfur atom substitutes a nonbridging oxygen in the DNA phosphodiester backbone. The initial sulfur mobilization from l-cysteine is catalyzed by the SspA cysteine desulfurase in the SspABCD-mediated DNA PT modification system. By determining the crystal structure of SspA, the study presents the molecular mechanism that SspA employs to recognize its cysteine substrate and PLP cofactor. To overcome the long distance (8.9 Å) between the catalytic Cys314 and the cysteine substrate, a conformational change occurs to bring Cys314 to the vicinity of the substrate, allowing for nucleophilic attack.

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Biochemical and structural characterization of oxygen-sensitive 2-thiouridine synthesis catalyzed by an iron-sulfur protein TtuA

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1615585114 ◽

2017 ◽

Vol 114 (19) ◽

pp. 4954-4959 ◽

Cited By ~ 19

Author(s):

Minghao Chen ◽

Shin-ichi Asai ◽

Shun Narai ◽

Shusuke Nambu ◽

Naoki Omura ◽

...

Keyword(s):

Crystal Structure ◽

Enzymatic Activity ◽

Molecular Mechanism ◽

Active Site ◽

Thermophilic Bacteria ◽

Oxygen Sensitive ◽

Sulfur Protein ◽

Combined Action

Two-thiouridine (s2U) at position 54 of transfer RNA (tRNA) is a posttranscriptional modification that enables thermophilic bacteria to survive in high-temperature environments. s2U is produced by the combined action of two proteins, 2-thiouridine synthetase TtuA and 2-thiouridine synthesis sulfur carrier protein TtuB, which act as a sulfur (S) transfer enzyme and a ubiquitin-like S donor, respectively. Despite the accumulation of biochemical data in vivo, the enzymatic activity by TtuA/TtuB has rarely been observed in vitro, which has hindered examination of the molecular mechanism of S transfer. Here we demonstrate by spectroscopic, biochemical, and crystal structure analyses that TtuA requires oxygen-labile [4Fe-4S]-type iron (Fe)-S clusters for its enzymatic activity, which explains the previously observed inactivation of this enzyme in vitro. The [4Fe-4S] cluster was coordinated by three highly conserved cysteine residues, and one of the Fe atoms was exposed to the active site. Furthermore, the crystal structure of the TtuA-TtuB complex was determined at a resolution of 2.5 Å, which clearly shows the S transfer of TtuB to tRNA using its C-terminal thiocarboxylate group. The active site of TtuA is connected to the outside by two channels, one occupied by TtuB and the other used for tRNA binding. Based on these observations, we propose a molecular mechanism of S transfer by TtuA using the ubiquitin-like S donor and the [4Fe-4S] cluster.

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Crystal Structure of Extended-Spectrum β-Lactamase Toho-1: Insights into the Molecular Mechanism for Catalytic Reaction and Substrate Specificity Expansion†,‡

Biochemistry ◽

10.1021/bi0342822 ◽

2003 ◽

Vol 42 (36) ◽

pp. 10634-10643 ◽

Cited By ~ 56

Author(s):

Akiko Shimizu Ibuka ◽

Yoshikazu Ishii ◽

Moreno Galleni ◽

Masaji Ishiguro ◽

Keizo Yamaguchi ◽

...

Keyword(s):

Crystal Structure ◽

Substrate Specificity ◽

Molecular Mechanism ◽

Catalytic Reaction ◽

Extended Spectrum

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Crystal structure of NADH:rubredoxin oxidoreductase from Clostridium acetobutylicum: A key component of the dioxygen scavenging system in obligatory anaerobes

Proteins Structure Function and Bioinformatics ◽

10.1002/prot.22650 ◽

2009 ◽

Vol 78 (4) ◽

pp. 1066-1070 ◽

Cited By ~ 2

Author(s):

Koji Nishikawa ◽

Yasuhito Shomura ◽

Shinji Kawasaki ◽

Youichi Niimura ◽

Yoshiki Higuchi

Keyword(s):

Crystal Structure ◽

Clostridium Acetobutylicum

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The crystal structure reveals the molecular mechanism of bifunctional 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II (Rv1415) fromMycobacterium tuberculosis

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444913011402 ◽

2013 ◽

Vol 69 (9) ◽

pp. 1633-1644 ◽

Cited By ~ 6

Author(s):

Mirage Singh ◽

Pankaj Kumar ◽

Savita Yadav ◽

Ruchi Gautam ◽

Nidhi Sharma ◽

...

Keyword(s):

Crystal Structure ◽

Molecular Mechanism ◽

Gtp Cyclohydrolase ◽

Gtp Cyclohydrolase Ii ◽

Phosphate Synthase

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Molecular Mechanism of SR Protein Kinase 1 Inhibition by the Herpes Virus Protein ICP27

mBio ◽

10.1128/mbio.02551-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 3

Author(s):

Richard B. Tunnicliffe ◽

William K. Hu ◽

Michele Y. Wu ◽

Colin Levy ◽

A. Paul Mould ◽

...

Keyword(s):

Crystal Structure ◽

Alternative Splicing ◽

Herpes Simplex ◽

Protein Kinase ◽

Molecular Mechanism ◽

Sr Proteins ◽

Sr Protein ◽

High Affinity ◽

Simplex Virus

ABSTRACT Serine-arginine (SR) protein kinase 1 (SRPK1) catalyzes the phosphorylation of SR proteins, which are a conserved family of splicing factors that contain a domain rich in arginine and serine repeats. SR proteins play important roles in constitutive pre-mRNA splicing and are also important regulators of alternative splicing. During herpes simplex virus infection, SRPK1 is inactivated and its cellular distribution is markedly altered by interaction with the viral protein ICP27, resulting in hypophosphorylation of SR proteins. Mutational analysis previously showed that the RGG box motif of ICP27 is required for interaction with SRPK1; however, the mechanism for the inhibition and the exact role of the RGG box was unknown. Here, we used solution nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC) to demonstrate that the isolated peptide comprising the RGG box of ICP27 binds to SRPK1 with high affinity, competing with a native substrate, the SR repeat region of SR protein SRSF1. We determined the crystal structure of the complex between SRPK1 and an RGG box peptide, which revealed that the viral peptide binds to the substrate docking groove, mimicking the interactions of SR repeats. Site-directed mutagenesis within the RGG box further confirmed the importance of selected arginine residues for interaction, relocalization, and inhibition of SRPK1 in vivo. Together these data reveal the molecular mechanism of the competitive inhibition of cellular SRPK1 by viral ICP27, which modulates SRPK1 activity. IMPORTANCE Serine arginine (SR) proteins are a family of mRNA regulatory proteins that can modulate spliceosome association with different splice sites and therefore regulate alternative splicing. Phosphorylation within SR proteins is necessary for splice-site recognition, and this is catalyzed by SR protein kinase 1 (SRPK1). The herpes simplex virus (HSV-1) protein ICP27 has been shown previously to interact with and downregulate SRPK1 activity in vivo; however, the molecular mechanism for this interaction and inhibition was unknown. Here, we demonstrate that the isolated peptide fragment of ICP27 containing RGG box binds to SRPK1 with high affinity, and competes with a native cellular substrate. Elucidation of the SRPK1-RGG box crystal structure further showed that a short palindromic RGRRRGR sequence binds in the substrate docking groove of SRPK1, mimicking the binding of SR repeats of substrates. These data reveal how the viral protein ICP27 inactivates SRPK1, promoting hypophosphorylation of proteins regulating splicing.

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Crystal structure and kinetic analyses of a hexameric form of (S)-3-hydroxybutyryl-CoA dehydrogenase from Clostridium acetobutylicum

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x18014814 ◽

2018 ◽

Vol 74 (11) ◽

pp. 733-740

Author(s):

Mihoko Takenoya ◽

Seiichi Taguchi ◽

Shunsuke Yajima

Keyword(s):

Crystal Structure ◽

Crystal Structures ◽

Biodegradable Polymers ◽

Clostridium Acetobutylicum ◽

Ralstonia Eutropha ◽

Homo Sapiens ◽

Gel Filtration ◽

Single Amino Acid ◽

Wild Type ◽

Key Enzyme

(S)-3-Hydroxybutyryl-CoA dehydrogenase (HBD) has been gaining increased attention recently as it is a key enzyme in the enantiomeric formation of (S)-3-hydroxybutyryl-CoA [(S)-3HB-CoA]. It converts acetoacetyl-CoA to (S)-3HB-CoA in the synthetic metabolic pathway. (S)-3HB-CoA is further modified to form (S)-3-hydroxybutyrate, which is a source of biodegradable polymers. During the course of a study to develop biodegradable polymers, attempts were made to determine the crystal structure of HBD from Clostridium acetobutylicum (CacHBD), and the crystal structures of both apo and NAD+-bound forms of CacHBD were determined. The crystals belonged to different space groups: P212121 and P21. However, both structures adopted a hexamer composed of three dimers in the asymmetric unit, and this oligomerization was additionally confirmed by gel-filtration column chromatography. Furthermore, to investigate the catalytic residues of CacHBD, the enzymatic activities of the wild type and of three single-amino-acid mutants were analyzed, in which the Ser, His and Asn residues that are conserved in the HBDs from C. acetobutylicum, C. butyricum and Ralstonia eutropha, as well as in the L-3-hydroxyacyl-CoA dehydrogenases from Homo sapiens and Escherichia coli, were substituted by alanines. The S117A and N188A mutants abolished the activity, while the H138A mutant showed a slightly lower K m value and a significantly lower k cat value than the wild type. Therefore, in combination with the crystal structures, it was shown that His138 is involved in catalysis and that Ser117 and Asn188 may be important for substrate recognition to place the keto group of the substrate in the correct position for reaction.

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