Asymmetry of the Active Site Loop Conformation between Subunits of Glutamate-1-semialdehyde Aminomutase in Solution

Glutamate-1-semialdehyde aminomutase (GSAM) is a dimeric, pyridoxal 5′-phosphate (PLP)- dependent enzyme catalysing in plants and some bacteria the isomerization of L-glutamate-1-semialdehyde to 5-aminolevulinate, a common precursor of chlorophyll, haem, coenzyme B12, and other tetrapyrrolic compounds. During the catalytic cycle, the coenzyme undergoes conversion from pyridoxamine 5′-phosphate (PMP) to PLP. The entrance of the catalytic site is protected by a loop that is believed to switch from an open to a closed conformation during catalysis. Crystallographic studies indicated that the structure of the mobile loop is related to the form of the cofactor bound to the active site, allowing for asymmetry within the dimer. Since no information on structural and functional asymmetry of the enzyme in solution is available in the literature, we investigated the active site accessibility by determining the cofactor fluorescence quenching of PMP- and PLP-GSAM forms. PLP-GSAM is partially quenched by potassium iodide, suggesting that at least one catalytic site is accessible to the anionic quencher and therefore confirming the asymmetry observed in the crystal structure. Iodide induces release of the cofactor from PMP-GSAM, apparently from only one catalytic site, therefore suggesting an asymmetry also in this form of the enzyme in solution, in contrast with the crystallographic data.

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Structural and Mutagenic Analysis of Metallo-β-Lactamase IMP-18

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00985-16 ◽

2016 ◽

Vol 60 (9) ◽

pp. 5521-5526 ◽

Cited By ~ 5

Author(s):

Takamitsu Furuyama ◽

Haruka Nonomura ◽

Yoshikazu Ishii ◽

Nancy D. Hanson ◽

Akiko Shimizu-Ibuka

Keyword(s):

Crystal Structure ◽

Active Site ◽

Kinetic Properties ◽

Amino Acid Residues ◽

Content Type ◽

Zinc Metalloenzymes ◽

Order Of Magnitude ◽

Sandwich Configuration ◽

The Relationship ◽

Loop Conformation

ABSTRACTIMP-type metallo-β-lactamases (MBLs) are exogenous zinc metalloenzymes that hydrolyze a broad range of β-lactams, including carbapenems. Here we report the crystal structure of IMP-18, an MBL cloned fromPseudomonas aeruginosa, at 2.0-Å resolution. The overall structure of IMP-18 resembles that of IMP-1, with an αβ/βα “folded sandwich” configuration, but the loop that covers the active site has a distinct conformation. The relationship between IMP-18's loop conformation and its kinetic properties was investigated by replacing the amino acid residues that can affect the loop conformation (Lys44, Thr50, and Ile69) in IMP-18 with those occupying the corresponding positions in the well-described enzyme IMP-1. The replacement of Thr50 with Pro considerably modified IMP-18's kinetic properties, specifically those pertaining to meropenem, with thekcat/Kmvalue increased by an order of magnitude. The results indicate that this is a key residue that defines the kinetic properties of IMP-type β-lactamases.

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Molecular Modeling and Virtual Screening of Molecular Inhibitors for Leptospiral Collagenase

10.21203/rs.3.rs-259091/v1 ◽

2021 ◽

Author(s):

Vikram Kumar ◽

Nagesh Srikaku ◽

Veeranarayanan Surya Aathmanathan ◽

Padikara K Satheeshkumar ◽

Madanan Gopalakrishnan Madathiparambil ◽

...

Keyword(s):

Crystal Structure ◽

Molecular Modeling ◽

Active Site ◽

Binding Sites ◽

Catalytic Site ◽

Simulation Studies ◽

C Terminus ◽

Ligand Binding Sites ◽

N Terminus ◽

The Stability

Abstract Collagenase is a virulence factor which facilitates the invasion of pathogenic Leptospira into the host. In the present study, the model of Leptopsiral collagenase was constructed by employing threading method with the crystal structure of collagenase G. Three ligand binding sites at N- terminus, catalytic site and C-terminus were predicted by Metapocket server. Among sixty seven inhibitors from the ChEBI and Zinc databases, Protohypericin is predicted as the best inhibitor since it binds at the catalytic site of Leptopsiral collagenase. Molecular dynamic simulation studies validated the stability of interaction between the active site of Leptospiral collagenase and Protohypericin. The docking and molecular simulation studies corroborated the potential of the ligand to curb leptospiral infection.

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Crystal Structure of Coproporphyrinogen Oxidase (CPO).

Blood ◽

10.1182/blood.v104.11.52.52 ◽

2004 ◽

Vol 104 (11) ◽

pp. 52-52

Author(s):

John D. Phillips ◽

Frank G. Whitby ◽

Christopher P. Hill ◽

James P. Kushner

Keyword(s):

Crystal Structure ◽

Conformational Change ◽

Active Site ◽

Anomalous Scattering ◽

Side Chains ◽

Beta Sheet ◽

Conserved Residues ◽

Dimer Interface ◽

Closed Conformation ◽

Hereditary Coproporphyria

Abstract CPO, an essential enzyme in the heme biosynthetic pathway, catalyzes the oxidative decarboxylation of two propionate side chains of coproporphyrinogen III to form vinyl groups in the product protoporphyrinogen IX. CPO mutations are responsible for the disease hereditary coproporphyria. All eukaryotes express a highly conserved, oxygen-dependant form of CPO. Yeast and human CPO have a sequence identity of 52% at the amino acid level. To probe the biochemical basis of catalytic activity and to determine the deleterious effects of clinically identified mutations, we determined the crystal structure of CPO from S. cerevisiae. This is the first reported structure of a eukaryotic CPO. A cDNA encoding CPO (Hem13p) was expressed in E. coli as a histidine tagged protein. Hem13p was purified and concentrated to 25 mg/ml and crystals were grown in the presence of 18% PEG 8000, 0.1 M HEPES, pH 7.5, 2% isopropanol, 0.2 M Na-acetate. The crystal structure was determined by optimized sulfur anomalous scattering and refined to a resolution of 2.0 Å. The protein folds into a novel structure featuring a central flat seven-stranded antiparallel beta-sheet flanked on each side with helices. The homodimeric structure of CPO is formed by a short isolated strand that forms a beta-ladder between the two monomers. Each monomer contains an active site formed between the flat beta-sheet and adjacent helices near the dimer interface. Many of the conserved residues are located at this interface. The deep active site cleft is lined by conserved residues and has been captured in an open and a closed conformation in two different crystal forms. The substrate cavity is completely buried in the closed conformation by an approximate 8 Å movement of a helix that forms a lid over the active site. The volume of the enclosed cavity precisely accommodates a modeled molecule of coproporphyrinogen III. The model indicates binding of two propionate side chains of coproporphyrinogen with two invariant arginines. The pyrrole nitrogens of the substrate are positioned to be coordinated by an invariant asparagine in a fashion similar to that described for uroporphyrinogen decarboxylase (EMBO J, 2003, 22: 6225–33). Nineteen point mutations have been described in association with hereditary coproporphyria and we have mapped these to the crystal structure. Only two mapped to the active site. Fourteen mutations were predicted to destabilize the protein and 4 of these occur at the dimer interface. Three mutations mapped to the solvent exposed surface of CPO. The crystal structure supports a model in which the enzymatic reaction occurs in an isolated cavity formed by a conformational change induced by substrate binding. The conformational change could create a binding site for molecular oxygen, the cofactor, and provide a mechanism to protect coproporphyrinogen III from inappropriate oxidation and the subsequent peroxidation of protoporphyrinogen IX.

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Crystal Structure of Bacillus subtilis α-Amylase in Complex with Acarbose

Journal of Bacteriology ◽

10.1128/jb.185.23.6981-6984.2003 ◽

2003 ◽

Vol 185 (23) ◽

pp. 6981-6984 ◽

Cited By ~ 20

Author(s):

Masayuki Kagawa ◽

Zui Fujimoto ◽

Mitsuru Momma ◽

Kenji Takase ◽

Hiroshi Mizuno

Keyword(s):

Crystal Structure ◽

Bacillus Subtilis ◽

Active Site ◽

Catalytic Site ◽

Catalytic Process ◽

Present Structure ◽

Initial Stage

ABSTRACT The crystal structure of Bacillus subtilis α-amylase, in complex with the pseudotetrasaccharide inhibitor acarbose, revealed an hexasaccharide in the active site as a result of transglycosylation. After comparison with the known structure of the catalytic-site mutant complexed with the native substrate maltopentaose, it is suggested that the present structure represents a mimic intermediate in the initial stage of the catalytic process.

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Dual-Function, Light Switchable Cobalt Catalysis via Active Site Metamorphosis

10.26434/chemrxiv.11353106.v1 ◽

2019 ◽

Author(s):

Enrico Bergamaschi ◽

Frédéric Beltran ◽

Christopher Teskey

Keyword(s):

Visible Light ◽

Active Site ◽

Catalytic Site ◽

Dual Function ◽

Catalytic Systems ◽

Hydride Complex ◽

Dual Functional ◽

Multiple Processes ◽

Olefin Migration

Switchable catalysis offers opportunities to control the rate or selectivity of a reaction via a stimulus such as pH or light. However, few examples of switchable catalytic systems that can facilitate multiple processes exist. Here we report a rare example of such dual-functional, switchable catalysis. Featuring an easily prepared, bench-stable cobalt(I) hydride complex in conjunction with pinacolborane, we can completely alter the reaction outcome between two widely employed transformations – olefin migration and hydroboration – with visible light as the sole trigger. This dichotomy arises from ligand photodissociation which leads to metamorphosis of the active catalytic site, resulting in divergent mechanistic pathways.

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