Interaction of the neutral amino acid transporter ASCT2 with basic amino acids

Glutamine transport across cell membranes is performed by a variety of transporters, including the alanine serine cysteine transporter 2 (ASCT2). The substrate-binding site of ASCT2 was proposed to be specific for small amino acids with neutral side chains, excluding basic substrates such as lysine. A series of competitive inhibitors of ASCT2 with low µM affinity were developed previously, on the basis of the 2,4-diaminobutyric acid (DAB) scaffold with a potential positive charge in the side chain. Therefore, we tested whether basic amino acids with side chains shorter than lysine can interact with the ASCT2 binding site. Molecular docking of L-1,3-diaminopropionic acid (L-DAP) and L-DAB suggested that these compounds bind to ASCT2. Consistent with this prediction, L-DAP and L-DAB, but not ornithine, lysine or D-DAP, elicited currents when applied to ASCT2-expressing cells. The currents were carried by anions and showed the hallmark properties of ASCT2 currents induced by transported substrates. The L-DAP response could be eliminated by a competitive ASCT2 inhibitor, suggesting that binding occurs at the substrate binding site. The KM for L-DAP was weakly voltage dependent. Furthermore, the pH dependence of the L-DAP response showed that the compound can bind in several protonation states. Together, these results suggest that the ASCT2 binding site is able to recognize L-amino acids with short, basic side chains, such as the L-DAP derivative β-N-methylamino-l-Alanine (BMAA), a well-studied neurotoxin. Our results expand the substrate specificity of ASCT2 to include amino acid substrates with positively charged side chains.

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Characterization of the extracellular poly(3-hydroxybutyrate) depolymerase ofComamonassp. and of its structural gene

Canadian Journal of Microbiology ◽

10.1139/m95-183 ◽

1995 ◽

Vol 41 (13) ◽

pp. 160-169 ◽

Cited By ~ 52

Author(s):

Dieter Jendrossek ◽

Martina Backhaus ◽

Meike Andermann

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Binding Site ◽

Catalytic Domain ◽

Substrate Binding ◽

Catalytic Triad ◽

Reading Frame ◽

Phb Depolymerase ◽

Substrate Binding Site

The poly(3-hydroxybutyrate) (PHB) depolymerase structural gene of Comamonas sp. (phaZCsp) was cloned in Escherichia coli and identified by halo formation on PHB-containing solid medium. The nucleotide sequence of a 1719 base pair MboI fragment was determined and contained one large open reading frame (ORF1, 1542 base pairs). This open reading frame encoded the precursor of the PHB depolymerase (514 amino acids; Mr, 53 095), and the deduced amino acid sequence was in agreement with the N-terminal amino acid sequence of the purified PHB depolymerase from amino acid 26 onwards. Analysis of the deduced amino acid sequence revealed a domain structure of the protein: a signal peptide that was 25 amino acids long was followed by a catalytic domain of about 300 amino acids, a fibronectin type III (Fn3) modul sequence, and a putative PHB-specific substrate-binding site. By comparison of the primary structure with that of other polyhydroxyalkanoate (PHA) depolymerases, the catalytic domain apparently contained a catalytic triad of serine, histidine, and aspartate. In addition, a conserved region resembling the oxyanion hole of lipases was present. The catalytic domain was linked to a C-terminal putative substrate-binding site by a sequence about 90 amino acids long resembling the Fn3 modul of fibronectin and other eukaryotic extracellular matrix proteins. A threonine-rich region, which was found in four of five PHA depolymerases of Pseudomonas lemoignei, was not present in the Comamonas sp. depolymerase. The similarities with and differences from other PHA depolymerases are discussed.Key words: biodegradable polymer, poly(3-hydroxybutyrate) depolymerase, serine hydrolase, catalytic triad, Comamonas sp., fibronectin type III modul, substrate-binding site.

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In-silico Investigation of the Interaction between Beta-class Glutathione S-Transferase and Five Antibiotics, namely; Ampicillin, Tetracycline, Chloramphenicol, Ciprofloxacin and Cephalexin

Journal of Applied Sciences and Environmental Management ◽

10.4314/jasem.v25i4.2 ◽

2021 ◽

Vol 25 (4) ◽

pp. 497-502

Author(s):

D. Shehu ◽

S Danlami ◽

M. Ya’u ◽

A. Babandi ◽

H.M. Yakasai ◽

...

Keyword(s):

Amino Acids ◽

Antibiotic Resistance ◽

Molecular Docking ◽

Binding Site ◽

Substrate Binding ◽

Docking Study ◽

Glutathione S Transferase ◽

Molecular Docking Study ◽

Substrate Binding Site ◽

Glutathione S Transferases

Glutathione s-transferases(GSTs) are enzymes involved in the conjugation and deactivation of various xenobiotics including drugs. Thisin-silico study was undertaken in order to investigate the interaction between beta-class glutathione s-transferase and five selected antibiotics, namely; ampicillin, tetracycline, chloramphenicol, ciprofloxacin and cephalexin using molecular docking study. RaptorX server was used to predict the amino acids involved at the binding sitewhile molecular docking study was employed in order to investigate the binding interactions.RaptorX predicted several amino acids which were different from the ones observed in molecular docking because of the variability in the substrate binding site of GSTs however, all the amino acids predicted by RaptorX were also found to be involved in the GSH binding.Lys107, Phe109, Ser110, Leu113, Trp114, His115 and Arg123, Leu168 were the amino acids involved in the binding of various antibiotics to the substrate binding site of the protein while Ala9, Cys10, Leu32, Tyr51, Val52, Pro53, Glu65 and Ala66were involved in the binding of the co-substrate GSH to the binding site of the protein. The results indicated that all the antibiotics showed a good binding affinity with the beta class GST and are therefore capable of deactivating the drugs. With these, finding a beta class GST inhibitors alongside antibiotics during a treatment of diseases will be of beneficial in the current fight against antibiotic resistance.

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Effects of Substrate-Binding Site Residues on the Biochemical Properties of a Tau Class Glutathione S-Transferase from Oryza sativa

Genes ◽

10.3390/genes11010025 ◽

2019 ◽

Vol 11 (1) ◽

pp. 25 ◽

Cited By ~ 2

Author(s):

Xue Yang ◽

Jinchi Wei ◽

Zhihai Wu ◽

Jie Gao

Keyword(s):

Amino Acids ◽

Enzyme Activity ◽

Abiotic Stress ◽

Binding Site ◽

Stress Responses ◽

Stress Resistance ◽

Substrate Binding ◽

Biochemical Properties ◽

Biotic And Abiotic Stress ◽

Substrate Binding Site

Glutathione S-transferases (GSTs)—an especially plant-specific tau class of GSTs—are key enzymes involved in biotic and abiotic stress responses. To improve the stress resistance of crops via the genetic modification of GSTs, we predicted the amino acids present in the GSH binding site (G-site) and hydrophobic substrate-binding site (H-site) of OsGSTU17, a tau class GST in rice. We then examined the enzyme activity, substrate specificity, enzyme kinetics and thermodynamic stability of the mutant enzymes. Our results showed that the hydrogen bonds between Lys42, Val56, Glu68, and Ser69 of the G-site and glutathione were essential for enzyme activity and thermal stability. The hydrophobic side chains of amino acids of the H-site contributed to enzyme activity toward 4-nitrobenzyl chloride but had an inhibitory effect on enzyme activity toward 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide. Different amino acids of the H-site had different effects on enzyme activity toward a different substrate, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Moreover, Leu112 and Phe162 were found to inhibit the catalytic efficiency of OsGSTU17 to 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, while Pro16, Leu112, and Trp165 contributed to structural stability. The results of this research enhance the understanding of the relationship between the structure and function of tau class GSTs to improve the abiotic stress resistance of crops.

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N‐Linoleyl amino acids as chiral probes for the substrate binding site of soybean lipoxygenase‐1

The FASEB Journal ◽

10.1096/fasebj.21.5.a276-d ◽

2007 ◽

Vol 21 (5) ◽

Author(s):

Charles H. Clapp ◽

Justin Pachuski ◽

Kathleen A. Bishop ◽

Megan M. Young

Keyword(s):

Amino Acids ◽

Binding Site ◽

Substrate Binding ◽

Soybean Lipoxygenase ◽

Substrate Binding Site

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Identification of an amino acid residue involved in the substrate-binding site of rat liver uricase by site-directed mutagenesis

Biochemical and Biophysical Research Communications ◽

10.1016/s0006-291x(05)81464-0 ◽

1992 ◽

Vol 187 (1) ◽

pp. 101-107 ◽

Cited By ~ 4

Author(s):

Masaki Ito ◽

Seiya Kato ◽

Masamichi Nakamura ◽

Go Mitiko ◽

Yasuyuki Takagi

Keyword(s):

Amino Acid ◽

Binding Site ◽

Rat Liver ◽

Amino Acid Residue ◽

Substrate Binding ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Substrate Binding Site

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Crystal structures of hydroxymethylbilane synthase complexed with a substrate analog: a single substrate-binding site for four consecutive condensation steps

Biochemical Journal ◽

10.1042/bcj20200996 ◽

2021 ◽

Vol 478 (5) ◽

pp. 1023-1042

Author(s):

Hideaki Sato ◽

Masakazu Sugishima ◽

Mai Tsukaguchi ◽

Takahiro Masuko ◽

Mikuru Iijima ◽

...

Keyword(s):

Binding Site ◽

Substrate Binding ◽

Thermal Fluctuation ◽

Crystal Structure Analysis ◽

Dynamics Simulation ◽

Side Chains ◽

Amino Acid Residues ◽

Substrate Binding Site ◽

Cofactor Binding ◽

Single Substrate

Hydroxymethylbilane synthase (HMBS), which is involved in the heme biosynthesis pathway, has a dipyrromethane cofactor and combines four porphobilinogen (PBG) molecules to form a linear tetrapyrrole, hydroxymethylbilane. Enzyme kinetic study of human HMBS using a PBG-derivative, 2-iodoporphobilinogen (2-I-PBG), exhibited noncompetitive inhibition with the inhibition constant being 5.4 ± 0.3 µM. To elucidate the reaction mechanism of HMBS in detail, crystal structure analysis of 2-I-PBG-bound holo-HMBS and its reaction intermediate possessing two PBG molecules (ES2), and inhibitor-free ES2 was performed at 2.40, 2.31, and 1.79 Å resolution, respectively. Their overall structures are similar to that of inhibitor-free holo-HMBS, and the differences are limited near the active site. In both 2-I-PBG-bound structures, 2-I-PBG is located near the terminus of the cofactor or the tetrapyrrole chain. The propionate group of 2-I-PBG interacts with the side chain of Arg173, and its acetate group is associated with the side chains of Arg26 and Ser28. Furthermore, the aminomethyl group and pyrrole nitrogen of 2-I-PBG form hydrogen bonds with the side chains of Gln34 and Asp99, respectively. These amino acid residues form a single substrate-binding site, where each of the four PBG molecules covalently binds to the cofactor (or oligopyrrole chain) consecutively, ultimately forming a hexapyrrole chain. Molecular dynamics simulation of the ES2 intermediate suggested that the thermal fluctuation of the lid and cofactor-binding loops causes substrate recruitment and oligopyrrole chain shift needed for consecutive condensation. Finally, the hexapyrrole chain is hydrolyzed self-catalytically to produce hydroxymethylbilane.

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Identification of an Amino Acid Residue That Lies between the Exofacial Vestibule and Exofacial Substrate-binding Site of the Glut1 Sugar Permeation Pathway

Journal of Biological Chemistry ◽

10.1074/jbc.272.48.30141 ◽

1997 ◽

Vol 272 (48) ◽

pp. 30141-30146 ◽

Cited By ~ 38

Author(s):

Mike Mueckler ◽

Carol Makepeace

Keyword(s):

Amino Acid ◽

Binding Site ◽

Amino Acid Residue ◽

Substrate Binding ◽

Substrate Binding Site

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Loss of Function in Zeaxanthin Epoxidase of Dunaliella tertiolecta Caused by a Single Amino Acid Mutation within the Substrate-Binding Site

Marine Drugs ◽

10.3390/md16110418 ◽

2018 ◽

Vol 16 (11) ◽

pp. 418 ◽

Cited By ~ 2

Author(s):

Minjae Kim ◽

Jisu Kang ◽

Yongsoo Kang ◽

Beom Kang ◽

EonSeon Jin

Keyword(s):

Amino Acid ◽

Binding Site ◽

Conformational Change ◽

Substrate Binding ◽

Single Amino Acid ◽

Wild Type ◽

Amino Acid Mutation ◽

Zeaxanthin Epoxidase ◽

Substrate Binding Site ◽

Single Amino Acid Mutation

The zea1 mutant of marine microalga Dunaliella tertiolecta accumulates zeaxanthin under normal growth conditions, and its phenotype has been speculated to be related to zeaxanthin epoxidase (ZEP). In this study, we isolated the ZEP gene from both wild-type D. tertiolecta and the mutant. We found that the zea1 mutant has a point mutation of the 1337th nucleotide of the ZEP sequence (a change from guanine to adenine), resulting in a change of glycine to aspartate in a highly conserved region in the catalytic domain. Similar expression levels of ZEP mRNA and protein in both wild-type and zea1 were confirmed by using qRT-PCR and western blot analysis, respectively. Additionally, the enzyme activity analysis of ZEPs in the presence of cofactors showed that the inactivation of ZEP in zea1 was not caused by deficiency in the levels of cofactors. From the predicted three-dimensional ZEP structure of zea1, we observed a conformational change on the substrate-binding site in the ZEP. A comparative analysis of the ZEP structures suggested that the conformational change induced by a single amino acid mutation might impact the interaction between the substrate and substrate-binding site, resulting in loss of zeaxanthin epoxidase function.

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Discrimination of the prochiral hydrogens at the C-2 position of n-alkanes by the methane/ammonia monooxygenase family proteins

Organic & Biomolecular Chemistry ◽

10.1039/c5ob00640f ◽

2015 ◽

Vol 13 (30) ◽

pp. 8261-8270 ◽

Cited By ~ 2

Author(s):

Akimitsu Miyaji ◽

Teppei Miyoshi ◽

Ken Motokura ◽

Toshihide Baba

Keyword(s):

Amino Acid ◽

Binding Site ◽

Substrate Binding ◽

Amino Acid Residues ◽

Ammonia Monooxygenase ◽

Substrate Binding Site ◽

Copper Site ◽

The Family ◽

Discriminating Ability

The substrate binding site of AMO/pMMO family proteins can discriminate between the prochiral hydrogens at the C-2 position ofn-alkanes. We predict that at least one of the three amino acid residues at the di-copper site affects the discriminating ability of the family proteins.

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