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

2018 ◽  
Vol 102 (4) ◽  
pp. 1737-1747 ◽  
Author(s):  
Yunhan Hsu ◽  
Hirohiko Koizumi ◽  
Masato Otagiri ◽  
Shigeharu Moriya ◽  
Manabu Arioka
Keyword(s):  
Substrate Binding ◽  
Critical Role ◽  
Termite Hindgut

scholarly journals Functional role of residues involved in substrate binding of human 4-hydroxyphenylpyruvate dioxygenase

2021 ◽  
Author(s):  
Chih-Wei Huang ◽  
Chi-Ching Hwang ◽  
Yung-Lung Chang ◽  
Jen-Tzu Liu ◽  
Sheng-Peng Wu ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Functional Role ◽  
Substrate Binding ◽  
Critical Role ◽  
Intermediate Formation ◽  
Hydroxyl Group ◽  
Double Mutation ◽  
Closed Conformation ◽  
Bond Network

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.

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

2009 ◽  
Vol 421 (2) ◽  
pp. 231-241 ◽  
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.

scholarly journals Insight into the mechanism of H+-coupled nucleobase transport

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.

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

2019 ◽  
Vol 294 (37) ◽  
pp. 13800-13810 ◽  
Author(s):  
Xuan Fang ◽  
Jerzy Osipiuk ◽  
Srinivas Chakravarthy ◽  
Ming Yuan ◽  
William M. Menzer ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Substrate Binding ◽  
Critical Role ◽  
Conserved Residue

scholarly journals The Roles of SPOP in DNA Damage Response and DNA Replication

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.

scholarly journals Spontaneous Phase Separation of Cocultured Cell Mixtures In vitro

2017 ◽  
Author(s):  
Sebastian V. Hadjiantoniou ◽  
Maxime Leblanc-Latour ◽  
Maxime Ignacio ◽  
Cory S. Lefevbre ◽  
Gary W. Slater ◽  
...  
Keyword(s):  
Phase Separation ◽  
Expression Profiles ◽  
Substrate Binding ◽  
Critical Role ◽  
Cellular Adhesion ◽  
Binding Energies ◽  
Cell Substrate ◽  
Spontaneous Phase ◽  
Cell Cell

ABSTRACTDuring Embryogenesis, cells undergo constant organizational remodelling. Biochemical and biophysical guidance cues act in tandem to guide migration and morphogenesis into distinct cellular patterns. It has been shown that various cell types will express different configurations of cellular adhesion molecules known as cadherins and integrins. Cocultured in vitro experiments have focused on revealing the extensive genetic expression profiles that modulate embryogenesis whilst overlooking the physical cell-cell and cell-substrate interactions that influence organization. We demonstrate that NIH3T3 and MDCK cells undergo a spontaneous phase separation when cocultured in vitro and that this phenomenon occurs through purely physical binding energies. A Monte Carlo simulation model of a mixture of cells with different cell-cell and cell-substrate binding energies reveals that the spontaneous phase separation occurs due to the minimization of interfacial free energy within the system. Cell-cell and cell-substrate binding plays a critical role in cell organization and is capable of phase separating different populations of cells in vitro.

scholarly journals The crystal structure of dGTPase reveals the molecular basis of dGTP selectivity

2019 ◽  
Vol 116 (19) ◽  
pp. 9333-9339 ◽  
Author(s):  
Christopher O. Barnes ◽  
Ying Wu ◽  
Jinhu Song ◽  
Guowu Lin ◽  
Elizabeth L. Baxter ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Substrate Binding ◽  
Critical Role ◽  
Free Ligand ◽  
Binding Pocket ◽  
Allosteric Site ◽  
Long Distance ◽  
Cellular Survival ◽  
Base Specificity

Deoxynucleotide triphosphohydrolases (dNTPases) play a critical role in cellular survival and DNA replication through the proper maintenance of cellular dNTP pools. While the vast majority of these enzymes display broad activity toward canonical dNTPs, such as the dNTPase SAMHD1 that blocks reverse transcription of retroviruses in macrophages by maintaining dNTP pools at low levels, Escherichia coli (Ec)-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. However, the mechanism behind dGTP selectivity is unclear. Here we present the free-, ligand (dGTP)- and inhibitor (GTP)-bound structures of hexameric Ec-dGTPase, including an X-ray free-electron laser structure of the free Ec-dGTPase enzyme to 3.2 Å. To obtain this structure, we developed a method that applied UV-fluorescence microscopy, video analysis, and highly automated goniometer-based instrumentation to map and rapidly position individual crystals randomly located on fixed target holders, resulting in the highest indexing rates observed for a serial femtosecond crystallography experiment. Our structures show a highly dynamic active site where conformational changes are coupled to substrate (dGTP), but not inhibitor binding, since GTP locks dGTPase in its apo- form. Moreover, despite no sequence homology, Ec-dGTPase and SAMHD1 share similar active-site and HD motif architectures; however, Ec-dGTPase residues at the end of the substrate-binding pocket mimic Watson–Crick interactions providing guanine base specificity, while a 7-Å cleft separates SAMHD1 residues from dNTP bases, abolishing nucleotide-type discrimination. Furthermore, the structures shed light on the mechanism by which long distance binding (25 Å) of single-stranded DNA in an allosteric site primes the active site by conformationally “opening” a tyrosine gate allowing enhanced substrate binding.

scholarly journals Norovirus Protease Structure and Antivirals Development

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2069
Author(s):  
Boyang Zhao ◽  
Liya Hu ◽  
Yongcheng Song ◽  
Ketki Patil ◽  
Sasirekha Ramani ◽  
...  
Keyword(s):  
Protease Inhibitors ◽  
Active Site ◽  
Substrate Binding ◽  
Critical Role ◽  
Conformational Flexibility ◽  
Current Status ◽  
Valuable Insight ◽  
Design And Development ◽  
Structural Differences ◽  
Binding Pockets

Human norovirus (HuNoV) infection is a global health and economic burden. Currently, there are no licensed HuNoV vaccines or antiviral drugs available. The protease encoded by the HuNoV genome plays a critical role in virus replication by cleaving the polyprotein and is an excellent target for developing small-molecule inhibitors. The current strategy for developing HuNoV protease inhibitors is by targeting the enzyme’s active site and designing inhibitors that bind to the substrate-binding pockets located near the active site. However, subtle differential conformational flexibility in response to the different substrates in the polyprotein and structural differences in the active site and substrate-binding pockets across different genogroups, hamper the development of effective broad-spectrum inhibitors. A comparative analysis of the available HuNoV protease structures may provide valuable insight for identifying novel strategies for the design and development of such inhibitors. The goal of this review is to provide such analysis together with an overview of the current status of the design and development of HuNoV protease inhibitors.

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