Characterization of the active site and insight into the binding mode of the anti-angiogenesis agent fumagillin to the manganese(II)-loaded methionyl aminopeptidase from Escherichia coli

Abstract The heterotrimeric electron-bifurcating [FeFe] hydrogenase (HydABC) from Thermotoga maritima (Tm) couples the endergonic reduction of protons (H+) by dihydronicotinamide adenine dinucleotide (NADH) (∆G0 ≈ 18 kJ mol−1) to the exergonic reduction of H+ by reduced ferredoxin (Fdred) (∆G0 ≈ − 16 kJ mol−1). The specific mechanism by which HydABC functions is not understood. In the current study, we describe the biochemical and spectroscopic characterization of TmHydABC recombinantly produced in Escherichia coli and artificially maturated with a synthetic diiron cofactor. We found that TmHydABC catalyzed the hydrogen (H2)-dependent reduction of nicotinamide adenine dinucleotide (NAD+) in the presence of oxidized ferredoxin (Fdox) at a rate of ≈17 μmol NADH min−1 mg−1. Our data suggest that only one flavin is present in the enzyme and is not likely to be the site of electron bifurcation. FTIR and EPR spectroscopy, as well as FTIR spectroelectrochemistry, demonstrated that the active site for H2 conversion, the H-cluster, in TmHydABC behaves essentially the same as in prototypical [FeFe] hydrogenases, and is most likely also not the site of electron bifurcation. The implications of these results are discussed with respect to the current hypotheses on the electron bifurcation mechanism of [FeFe] hydrogenases. Overall, the results provide insight into the electron-bifurcating mechanism and present a well-defined system for further investigations of this fascinating class of [FeFe] hydrogenases. Graphic abstract

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Molecular Modeling and Active Site Binding Mode Characterization of Aspartate β-Semialdehyde Dehydrogenase Family

Molecular Informatics ◽

10.1002/minf.201200128 ◽

2013 ◽

Vol 32 (4) ◽

pp. 377-383 ◽

Cited By ~ 6

Author(s):

Rajender Kumar ◽

Prabha Garg

Keyword(s):

Molecular Modeling ◽

Active Site ◽

Binding Mode ◽

Semialdehyde Dehydrogenase ◽

Site Binding

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Flavonoids as Putative Epi-Modulators: Insight into Their Binding Mode with BRD4 Bromodomains Using Molecular Docking and Dynamics

Biomolecules ◽

10.3390/biom8030061 ◽

2018 ◽

Vol 8 (3) ◽

pp. 61 ◽

Cited By ~ 9

Author(s):

Fernando Prieto-Martínez ◽

José Medina-Franco

Keyword(s):

Kinase Inhibitors ◽

Binding Mode ◽

Binding Modes ◽

Ligand Interaction ◽

Bet Inhibitors ◽

Protein Ligand Interaction ◽

Brd4 Inhibition ◽

Dynamics Simulations ◽

Insight Into

Flavonoids are widely recognized as natural polydrugs, given their anti-inflammatory, antioxidant, sedative, and antineoplastic activities. Recently, different studies showed that flavonoids have the potential to inhibit bromodomain and extraterminal (BET) bromodomains. Previous reports suggested that flavonoids bind between the Z and A loops of the bromodomain (ZA channel) due to their orientation and interactions with P86, V87, L92, L94, and N140. Herein, a comprehensive characterization of the binding modes of fisetin and the biflavonoid, amentoflavone, is discussed. To this end, both compounds were docked with BET bromodomain 4 (BRD4) using four docking programs. The results were post-processed with protein–ligand interaction fingerprints. To gain further insight into the binding mode of the two natural products, the docking results were further analyzed with molecular dynamics simulations. The results showed that amentoflavone makes numerous contacts in the ZA channel, as previously described for flavonoids and kinase inhibitors. It was also found that amentoflavone can potentially make contacts with non-canonical residues for BET inhibition. Most of these contacts were not observed with fisetin. Based on these results, amentoflavone was experimentally tested for BRD4 inhibition, showing activity in the micromolar range. This work may serve as the basis for scaffold optimization and the further characterization of flavonoids as BET inhibitors.

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Structural and Functional Characterization of the Human Thymidylate Synthase (hTS) Interface Variant R175C, New Perspectives for the Development of hTS Inhibitors

Molecules ◽

10.3390/molecules24071362 ◽

2019 ◽

Vol 24 (7) ◽

pp. 1362

Author(s):

Cecilia Pozzi ◽

Stefania Ferrari ◽

Rosaria Luciani ◽

Maria Costi ◽

Stefano Mangani

Keyword(s):

Thymidylate Synthase ◽

Active Site ◽

Functional Characterization ◽

Binding Mode ◽

Anticancer Chemotherapy ◽

Innovative Strategies ◽

X Ray Crystallography ◽

Site Targeting ◽

New Strategies

Human thymidylate synthase (hTS) is pivotal for cell survival and proliferation, indeed it provides the only synthetic source of dTMP, required for DNA biosynthesis. hTS represents a validated target for anticancer chemotherapy. However, active site-targeting drugs towards hTS have limitations connected to the onset of resistance. Thus, new strategies have to be applied to effectively target hTS without inducing resistance in cancer cells. Here, we report the generation and the functional and structural characterization of a new hTS interface variant in which Arg175 is replaced by a cysteine. Arg175 is located at the interface of the hTS obligate homodimer and protrudes inside the active site of the partner subunit, in which it provides a fundamental contribution for substrate binding. Indeed, the R175C variant results catalytically inactive. The introduction of a cysteine at the dimer interface is functional for development of new hTS inhibitors through innovative strategies, such as the tethering approach. Structural analysis, performed through X-ray crystallography, has revealed that a cofactor derivative is entrapped inside the catalytic cavity of the hTS R175C variant. The peculiar binding mode of the cofactor analogue suggests new clues exploitable for the design of new hTS inhibitors.

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Comparison of the structural and kinetic properties of the cytochrome c nitrite reductases from Escherichia coli, Wolinella succinogenes, Sulfurospirillum deleyianum and Desulfovibrio desulfuricans

Biochemical Society Transactions ◽

10.1042/bst0340143 ◽

2006 ◽

Vol 34 (1) ◽

pp. 143-145 ◽

Cited By ~ 17

Author(s):

T.A. Clarke ◽

A.M. Hemmings ◽

B. Burlat ◽

J.N. Butt ◽

J.A. Cole ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Active Site ◽

Desulfovibrio Desulfuricans ◽

Amino Acid Sequences ◽

Kinetic Properties ◽

Wolinella Succinogenes ◽

Nitrite Reductases ◽

Sulphite Reductase

The recent crystallographic characterization of NrfAs from Sulfurospirillum deleyianum, Wolinella succinogenes, Escherichia coli and Desulfovibrio desulfuricans allows structurally conserved regions to be identified. Comparison of nitrite and sulphite reductase activities from different bacteria shows that the relative activities vary according to organism. By comparison of both amino acid sequences and structures, differences can be identified in the monomer–monomer interface and the active-site channel; these differences could be responsible for the observed variance in substrate activity and indicate that subtle changes in the NrfA structure may optimize the enzyme for different roles.

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Characterization of Escherichia coli thioredoxins with altered active site residues

Biochemistry ◽

10.1021/bi00467a016 ◽

1990 ◽

Vol 29 (15) ◽

pp. 3701-3709 ◽

Cited By ~ 44

Author(s):

Florence K. Gleason ◽

Chang Jin Lim ◽

Maryam Gerami-Nejad ◽

James A. Fuchs

Keyword(s):

Escherichia Coli ◽

Active Site ◽

Active Site Residues

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Characterization of Two New Aminopeptidases in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.187.11.3671-3677.2005 ◽

2005 ◽

Vol 187 (11) ◽

pp. 3671-3677 ◽

Cited By ~ 9

Author(s):

Yu Zheng ◽

Richard J. Roberts ◽

Simon Kasif ◽

Chudi Guan

Keyword(s):

Escherichia Coli ◽

Stop Codon ◽

Bacterial Species ◽

Start Codon ◽

Aminopeptidase Activity ◽

Peptide Substrates ◽

E Coli ◽

Terminal Domain ◽

Methionyl Aminopeptidase

ABSTRACT Two genes in the Escherichia coli genome, ypdE and ypdF, have been cloned and expressed, and their products have been purified. YpdF is shown to be a metalloenzyme with Xaa-Pro aminopeptidase activity and limited methionine aminopeptidase activity. Genes homologous to ypdF are widely distributed in bacterial species. The unique feature in the sequences of the products of these genes is a conserved C-terminal domain and a variable N-terminal domain. Full or partial deletion of the N terminus in YpdF leads to the loss of enzymatic activity. The conserved C-terminal domain is homologous to that of the methionyl aminopeptidase (encoded by map) in E. coli. However, YpdF and Map differ in their preference for the amino acid next to the initial methionine in the peptide substrates. The implication of this difference is discussed. ypdE is the immediate downstream gene of ypdF, and its start codon overlaps with the stop codon of ypdF by 1 base. YpdE is shown to be a metalloaminopeptidase and has a broad exoaminopeptidase activity.

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Structural characterization of a nonhydrolyzing UDP-GlcNAc 2-epimerase from Neisseria meningitidis serogroup A

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x20013680 ◽

2020 ◽

Vol 76 (11) ◽

pp. 557-567

Author(s):

Nicholas K. Hurlburt ◽

Jasper Guan ◽

Hoonsan Ong ◽

Hai Yu ◽

Xi Chen ◽

...

Keyword(s):

Neisseria Meningitidis ◽

Active Site ◽

Pathogenic Bacteria ◽

Allosteric Site ◽

Additional Insight ◽

Large Conformational Change ◽

Surface Polysaccharides ◽

Important Intermediate ◽

Insight Into

Bacterial nonhydrolyzing UDP-N-acetylglucosamine 2-epimerases catalyze the reversible interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmannosamine (UDP-ManNAc). UDP-ManNAc is an important intermediate in the biosynthesis of certain cell-surface polysaccharides, including those in some pathogenic bacteria, such as Neisseria meningitidis and Streptococcus pneumoniae. Many of these epimerases are allosterically regulated by UDP-GlcNAc, which binds adjacent to the active site and is required to initiate UDP-ManNAc epimerization. Here, two crystal structures of UDP-N-acetylglucosamine 2-epimerase from Neisseria meningitidis serogroup A (NmSacA) are presented. One crystal structure is of the substrate-free enzyme, while the other structure contains UDP-GlcNAc substrate bound to the active site. Both structures form dimers as seen in similar epimerases, and substrate binding to the active site induces a large conformational change in which two Rossmann-like domains clamp down on the substrate. Unlike other epimerases, NmSacA does not require UDP-GlcNAc to instigate the epimerization of UDP-ManNAc, although UDP-GlcNAc was found to enhance the rate of epimerization. In spite of the conservation of residues involved in binding the allosteric UDP-GlcNAc observed in similar UDP-GlcNAc 2-epimerases, the structures presented here do not contain UDP-GlcNAc bound in the allosteric site. These structural results provide additional insight into the mechanism and regulation of this critical enzyme and improve the structural understanding of the ability of NmSacA to epimerize modified substrates.

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