Conserved residue His-257 of Vibrio cholerae flavin transferase ApbE plays a critical role in substrate binding and catalysis

4-Hydroxylphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxylphenylpyruvate (HPP) to homogentisate, the important step for tyrosine catabolism. Comparison of the structure of human HPPD with the substrate-bound structure of A. thaliana HPPD revealed notably different orientations of the C-terminal helix. This helix performed as a closed conformation in human enzyme. Simulation revealed a different substrate-binding mode in which the carboxyl group of HPP interacted by a H-bond network formed by Gln334, Glu349 (the metal-binding ligand), and Asn363 (in the C-terminal helix). The 4-hydroxyl group of HPP interacted with Gln251 and Gln265. The relative activity and substrate-binding affinity were preserved for the Q334A mutant, implying the alternative role of Asn363 for HPP binding and catalysis. The reduction in kcat/Km of the Asn363 mutants confirmed the critical role in catalysis. Compared to the N363A mutant, the dramatic reduction in the Kd and thermal stability of the N363D mutant implies the side-chain effect in the hinge region rotation of the C-terminal helix. The activity and binding affinity were not recovered by double mutation; however, the 4-hydroxyphenylacetate intermediate formation by the uncoupled reaction of Q334N/N363Q and Q334A/N363D mutants indicated the importance of the H-bond network in the electrophilic reaction. These results highlight the functional role of the H-bond network in a closed conformation of the C-terminal helix to stabilize the bound substrate. The extremely low activity and reduction in Q251E’s Kd suggest that interaction coupled with the H-bond network is crucial to locate the substrate for nucleophilic reaction.

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Trp residue at subsite − 5 plays a critical role in the substrate binding of two protistan GH26 β-mannanases from a termite hindgut

Applied Microbiology and Biotechnology ◽

10.1007/s00253-017-8726-2 ◽

2018 ◽

Vol 102 (4) ◽

pp. 1737-1747 ◽

Cited By ~ 6

Author(s):

Yunhan Hsu ◽

Hirohiko Koizumi ◽

Masato Otagiri ◽

Shigeharu Moriya ◽

Manabu Arioka

Keyword(s):

Substrate Binding ◽

Critical Role ◽

Termite Hindgut

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Functional characterization of LePGT1, a membrane-bound prenyltransferase involved in the geranylation of p-hydroxybenzoic acid

Biochemical Journal ◽

10.1042/bj20081968 ◽

2009 ◽

Vol 421 (2) ◽

pp. 231-241 ◽

Cited By ~ 33

Author(s):

Kazuaki Ohara ◽

Ayumu Muroya ◽

Nobuhiro Fukushima ◽

Kazufumi Yazaki

Keyword(s):

Molecular Model ◽

Substrate Binding ◽

Expression System ◽

Critical Role ◽

Functional Characterization ◽

Site Directed Mutagenesis ◽

Hydroxybenzoic Acid ◽

Membrane Bound

The AS-PT (aromatic substrate prenyltransferase) family plays a critical role in the biosynthesis of important quinone compounds such as ubiquinone and plastoquinone, although biochemical characterizations of AS-PTs have rarely been carried out because most members are membrane-bound enzymes with multiple transmembrane α-helices. PPTs [PHB (p-hydroxybenzoic acid) prenyltransferases] are a large subfamily of AS-PTs involved in ubiquinone and naphthoquinone biosynthesis. LePGT1 [Lithospermum erythrorhizon PHB geranyltransferase] is the regulatory enzyme for the biosynthesis of shikonin, a naphthoquinone pigment, and was utilized in the present study as a representative of membrane-type AS-PTs to clarify the function of this enzyme family at the molecular level. Site-directed mutagenesis of LePGT1 with a yeast expression system indicated three out of six conserved aspartate residues to be critical to the enzymatic activity. A detailed kinetic analysis of mutant enzymes revealed the amino acid residues responsible for substrate binding were also identified. Contrary to ubiquinone biosynthetic PPTs, such as UBIA in Escherichia coli which accepts many prenyl substrates of different chain lengths, LePGT1 can utilize only geranyl diphosphate as its prenyl substrate. Thus the substrate specificity was analysed using chimeric enzymes derived from LePGT1 and UBIA. In vitro and in vivo analyses of the chimeras suggested that the determinant region for this specificity was within 130 amino acids of the N-terminal. A 3D (three-dimensional) molecular model of the substrate-binding site consistent with these biochemical findings was generated.

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The Highly Conserved Bacterial RNase YbeY Is Essential in Vibrio cholerae, Playing a Critical Role in Virulence, Stress Regulation, and RNA Processing

PLoS Pathogens ◽

10.1371/journal.ppat.1004175 ◽

2014 ◽

Vol 10 (6) ◽

pp. e1004175 ◽

Cited By ~ 36

Author(s):

Maarten Vercruysse ◽

Caroline Köhrer ◽

Bryan W. Davies ◽

Markus F. F. Arnold ◽

John J. Mekalanos ◽

...

Keyword(s):

Vibrio Cholerae ◽

Rna Processing ◽

Critical Role ◽

Stress Regulation

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A Highly Conserved gp120 Inner Domain Residue Modulates Env Conformation and Trimer Stability

Journal of Virology ◽

10.1128/jvi.01068-16 ◽

2016 ◽

Vol 90 (19) ◽

pp. 8395-8409 ◽

Cited By ~ 12

Author(s):

Shilei Ding ◽

William D. Tolbert ◽

Jérémie Prévost ◽

Beatriz Pacheco ◽

Mathieu Coutu ◽

...

Keyword(s):

Chain Length ◽

Conformational Changes ◽

Viral Entry ◽

Critical Role ◽

Aliphatic Chain ◽

Compensatory Mutation ◽

Link Type ◽

Layer 2 ◽

Conserved Residue ◽

Hiv 1

ABSTRACTPrevious studies have shown that highly conserved residues in the inner domain of gp120 are required for HIV-1 envelope glycoprotein (Env) transitions to the CD4-bound conformation (A. Finzi, S. H. Xiang, B. Pacheco, L. Wang, J. Haight, et al., Mol Cell37:656–667, 2010,http://dx.doi.org/10.1016/j.molcel.2010.02.012; A. Desormeaux, M. Coutu, H. Medjahed, B. Pacheco, A. Herschhorn, et al., J Virol87:2549–2562, 2013,http://dx.doi.org/10.1128/JVI.03104-12). Moreover, W69, a highly conserved residue located at the interface between layer 1 and layer 2 of the inner domain, was recently shown to be important for efficient Env recognition by CD4-induced (CD4i) antibodies capable of potent antibody-dependent cellular cytotoxicity (W. D. Tolbert, N. Gohain, M. Veillette, J. P. Chapleau, C. Orlandi, et al., 2016, Structure24:697–709,http://dx.doi.org/10.1016/j.str.2016.03.005; S. Ding, M. Veillette, M. Coutu, J. Prevost, L. Scharf, et al., 2016, J Virol90:2127–2134,http://dx.doi.org/10.1128/JVI.02779-15). We evaluated the contribution of the hydrophobicity of W69 to conformational changes of Env by replacing it with a series of residues with aliphatic or aromatic side chains of decreasing chain length. We have found that the hydrophobicity of residue 69 is important for Env processing, CD4 binding, and its transition to the CD4-bound conformation. The most deleterious effect was observed when W69 was replaced with alanine or glycine residues. However, the functions lost due to W69 mutations could be progressively restored with amino acids of increasing aliphatic chain length and fully recovered with residues bearing an aromatic ring. Interestingly, poor CD4 binding of W69A could be fully restored by introducing a compensatory mutation within layer 2 (S115W). Structural studies of HIV-1 gp120 coreeW69A/S115W mutant bound to the CD4 peptide mimetic M48U1 and Fab of anti-cluster A antibody N60-i3 revealed no perturbations to the overall structure of the double mutant compared to the wild-type protein but identified higher mobility within the interface between layer 1 and layer 2, the bridging sheet region, and the CD4 binding site.IMPORTANCEHIV-1 Env transitions to the CD4-bound conformation are required for viral entry. Previous studies identified a highly conserved residue of the inner domain, W69, as being involved in these conformational transitions (A. Finzi, S. H. Xiang, B. Pacheco, L. Wang, J. Haight, et al., Mol Cell 37:656–667, 2010,http://dx.doi.org/10.1016/j.molcel.2010.02.012). Here, we show that W69, located at the interface between gp120 and gp41 in the PGT151-bound trimer, plays a critical role in the interprotomer signaling induced by CD4 binding. This new information might be useful in immunogen design.

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Insight into the mechanism of H+-coupled nucleobase transport

10.1101/2021.12.20.473561 ◽

2021 ◽

Author(s):

Jun Weng ◽

Xiaoming Zhou ◽

Pattama Wiriyasermkul ◽

Zhenning Ren ◽

Xiuwen Yan ◽

...

Keyword(s):

Ascorbic Acid ◽

Binding Site ◽

Substrate Binding ◽

Critical Role ◽

Binding Domain ◽

Purine Base ◽

Substrate Binding Site ◽

Functional Studies ◽

Dependent Transport ◽

Substrate Binding Domain

Members of the nucleobase/ascorbic acid transporter (NAT) gene family are found in all kingdoms of life. In mammals, the concentrative uptake of ascorbic acid (vitamin C) by members of the NAT family is driven by the Na+ gradient, while the uptake of nucleobases in bacteria is powered by the H+ gradient. Here we report the structure and function PurTCp, a NAT family member from Colwellia psychrerythraea. The structure of PurTCp was determined to 2.80 Å resolution by X-ray crystallography. PurTCp forms a homodimer and each protomer has 14 transmembrane segments folded into a substrate-binding domain (core domain) and an interface domain (gate domain) A purine base is present in the structure and defines the location of the substrate binding site. Functional studies reveal that PurTCp transports purines but not pyrimidines, and that purine binding and transport is dependent on the pH. Mutation of a conserved aspartate residue close to the substrate binding site reveals the critical role of this residue in H+-dependent transport of purines. Comparison of the PurTCp structure with transporters of the same structural fold suggests that rigid-body motions of the substrate-binding domain are central for substrate translocation across the membrane.

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A potential substrate binding pocket of BdcA plays a critical role in NADPH recognition and biofilm dispersal

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2018.02.143 ◽

2018 ◽

Vol 497 (3) ◽

pp. 863-868 ◽

Cited By ~ 1

Author(s):

Wen-Si Yang ◽

Yuan Hong ◽

Ying Zhang ◽

Da-Cheng Wang ◽

De-Feng Li ◽

...

Keyword(s):

Substrate Binding ◽

Critical Role ◽

Binding Pocket ◽

Substrate Binding Pocket ◽

Biofilm Dispersal ◽

Potential Substrate

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Revisiting the role of cholesterol in regulating the pore-formation mechanism of Vibrio cholerae cytolysin, a membrane-damaging β-barrel pore-forming toxin

Biochemical Journal ◽

10.1042/bcj20180387 ◽

2018 ◽

Vol 475 (19) ◽

pp. 3039-3055 ◽

Cited By ~ 5

Author(s):

Reema Kathuria ◽

Anish Kumar Mondal ◽

Rohan Sharma ◽

Samarjit Bhattacharyya ◽

Kausik Chattopadhyay

Keyword(s):

Vibrio Cholerae ◽

Formation Mechanism ◽

Pore Formation ◽

Critical Role ◽

Lipid Vesicles ◽

Membrane Lipid ◽

Human Erythrocytes ◽

Binding Ability ◽

Cholesterol Binding

Vibrio cholerae cytolysin (VCC) is a β-barrel pore-forming toxin with potent membrane-damaging cell-killing activity. Previous studies employing the model membranes of lipid vesicles (liposomes) have shown that pore formation by VCC requires the presence of cholesterol in the liposome membranes. However, the exact role of cholesterol in the mode of action of VCC still remains unclear. Most importantly, implication of cholesterol, if any, in regulating the pore-formation mechanism of VCC in the biomembranes of eukaryotic cells remains unexplored. Here, we show that the presence of cholesterol promotes the interaction of VCC with the membrane lipid bilayer, when non-lipid-dependent interactions are absent. However, in the case of biomembranes of human erythrocytes, where accessory interactions are available, cholesterol appears to play a less critical role in the binding step. Nevertheless, in the absence of an optimal level of membrane cholesterol in the human erythrocytes, membrane-bound fraction of the toxin remains trapped in the form of abortive oligomeric assembly, devoid of functional pore-forming activity. Our study also shows that VCC exhibits a prominent propensity to associate with the cholesterol-rich membrane micro-domains of human erythrocytes. Interestingly, mutation of the cholesterol-binding ability of VCC does not block association with the cholesterol-rich membrane micro-domains on human erythrocytes. Based on these results, we propose that the specific cholesterol-binding ability of VCC does not appear to dictate its association with the cholesterol-rich micro-domains on human erythrocytes. Rather, targeting of VCC toward the membrane micro-domains of human erythrocytes possibly acts to facilitate the cholesterol-dependent pore-formation mechanism of the toxin.

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The Roles of SPOP in DNA Damage Response and DNA Replication

International Journal of Molecular Sciences ◽

10.3390/ijms21197293 ◽

2020 ◽

Vol 21 (19) ◽

pp. 7293

Author(s):

Masashi Maekawa ◽

Shigeki Higashiyama

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Dna Damage Response ◽

Genome Stability ◽

Substrate Binding ◽

Critical Role ◽

Tumor Formation ◽

Missense Mutations ◽

Cellular Functions ◽

Damage Response

Speckle-type BTB/POZ protein (SPOP) is a substrate recognition receptor of the cullin-3 (CUL3)/RING type ubiquitin E3 complex. To date, approximately 30 proteins have been identified as ubiquitinated substrates of the CUL3/SPOP complex. Pathologically, missense mutations in the substrate-binding domain of SPOP have been found in prostate and endometrial cancers. Prostate and endometrial cancer-associated SPOP mutations lose and increase substrate-binding ability, respectively. Expression of these SPOP mutants, thus, causes aberrant turnovers of the substrate proteins, leading to tumor formation. Although the molecular properties of SPOP and its cancer-associated mutants have been intensively elucidated, their cellular functions remain unclear. Recently, a number of studies have uncovered the critical role of SPOP and its mutants in DNA damage response and DNA replication. In this review article, we summarize the physiological functions of SPOP as a “gatekeeper” of genome stability.

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