Identification of essential lysyl and cysteinyl residues, and the amino acid sequence at the substrate-binding site of retinal oxidase

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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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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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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The LAT1 amino acid transporter: substrate binding site and drug discovery

Biochimica et Biophysica Acta (BBA) - Bioenergetics ◽

10.1016/j.bbabio.2018.09.278 ◽

2018 ◽

Vol 1859 ◽

pp. e94

Author(s):

Cesare Indiveri ◽

Mariafrancesca Scalise ◽

Michele Galluccio ◽

Lara Console

Keyword(s):

Amino Acid ◽

Drug Discovery ◽

Binding Site ◽

Substrate Binding ◽

Amino Acid Transporter ◽

Substrate Binding Site ◽

Acid Transporter

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

Biochemical Journal ◽

10.1042/bcj20190859 ◽

2020 ◽

Vol 477 (8) ◽

pp. 1443-1457

Author(s):

Elias Ndaru ◽

Rachel-Ann A. Garibsingh ◽

Laura Zielewicz ◽

Avner Schlessinger ◽

Christof Grewer

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Binding Site ◽

Substrate Binding ◽

Ph Dependence ◽

Side Chains ◽

Substrate Binding Site ◽

Basic Amino Acids ◽

Voltage Dependent ◽

Diaminopropionic Acid

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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Enhancement of the latent 3-isopropylmalate dehydrogenase activity of promiscuous homoisocitrate dehydrogenase by directed evolution

Biochemical Journal ◽

10.1042/bj20101246 ◽

2010 ◽

Vol 431 (3) ◽

pp. 401-412 ◽

Cited By ~ 9

Author(s):

Yumewo Suzuki ◽

Kuniko Asada ◽

Junichi Miyazaki ◽

Takeo Tomita ◽

Tomohisa Kuzuyama ◽

...

Keyword(s):

Crystal Structure ◽

Amino Acid ◽

Binding Site ◽

Directed Evolution ◽

Substrate Binding ◽

Dna Shuffling ◽

Turnover Number ◽

Substrate Binding Site ◽

Km Value ◽

Isopropylmalate Dehydrogenase

HICDH (homoisocitrate dehydrogenase), which is involved in lysine biosynthesis through α-aminoadipate, is a paralogue of IPMDH [3-IPM (3-isopropylmalate) dehydrogenase], which is involved in leucine biosynthesis. TtHICDH (Thermus thermophilus HICDH) can recognize isocitrate, as well as homoisocitrate, as the substrate, and also shows IPMDH activity, although at a considerably decreased rate. In the present study, the promiscuous TtHICDH was evolved into an enzyme showing distinct IPMDH activity by directed evolution using a DNA-shuffling technique. Through five repeats of DNA shuffling/screening, variants that allowed Escherichia coli C600 (leuB−) to grow on a minimal medium in 2 days were obtained. One of the variants LR5–1, with eight amino acid replacements, was found to possess a 65-fold increased kcat/Km value for 3-IPM, compared with TtHICDH. Introduction of a single back-replacement H15Y change caused a further increase in the kcat/Km value and a partial recovery of the decreased thermotolerance of LR5–1. Site-directed mutagenesis revealed that most of the amino acid replacements found in LR5–1 effectively increased IPMDH activity; replacements around the substrate-binding site contributed to the improved recognition for 3-IPM, and other replacements at sites away from the substrate-binding site enhanced the turnover number for the IPMDH reaction. The crystal structure of LR5–1 was determined at 2.4 Å resolution and revealed that helix α4 was displaced in a manner suitable for recognition of the hydrophobic γ-moiety of 3-IPM. On the basis of the crystal structure, possible reasons for enhancement of the turnover number are discussed.

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The 5-ketofructose reductase of Gluconobacter sp. strain CHM43 is a novel class in the shikimate dehydrogenase family

Journal of Bacteriology ◽

10.1128/jb.00558-20 ◽

2021 ◽

Author(s):

Thuy Minh Nguyen ◽

Masaru Goto ◽

Shohei Noda ◽

Minenosuke Matsutani ◽

Yuki Hodoya ◽

...

Keyword(s):

Crystal Structure ◽

Amino Acid ◽

Binding Site ◽

Catalytic Mechanism ◽

Substrate Binding ◽

High Yield ◽

Strong Increase ◽

Amino Acid Residues ◽

Shikimate Dehydrogenase ◽

Substrate Binding Site

Gluconobacter sp. CHM43 oxidizes mannitol to fructose and then does fructose to 5-keto-D-fructose (5KF) in the periplasmic space. Since NADPH-dependent 5KF reductase was found in the soluble fraction of Gluconobacter spp., 5KF might be transported into the cytoplasm and metabolized. Here we identified the GLF_2050 gene as the kfr gene encoding 5KF reductase (KFR). A mutant strain devoid of the kfr gene showed lower KFR activity and no 5KF consumption. The crystal structure revealed that KFR is similar to NADP + -dependent shikimate dehydrogenase (SDH), which catalyzes the reversible NADP + -dependent oxidation of shikimate to 3-dehydroshikimate. We found that several amino acid residues in the putative substrate-binding site of KFR were different from those of SDH. Phylogenetic analyses revealed that only a subclass in the SDH family containing KFR conserved such a unique substrate-binding site. We constructed KFR derivatives with amino acid substitutions, including replacement of Asn21 in the substrate-binding site with Ser that is found in SDH. The KFR-N21S derivative showed a strong increase in the K M value for 5KF, but a higher shikimate oxidation activity than wild-type KFR, suggesting that Asn21 is important for 5KF binding. In addition, the conserved catalytic dyad Lys72 and Asp108 were individually substituted for Asn. The K72N and D108N derivatives showed only negligible activities without a dramatic change in the K M value for 5KF, suggesting a similar catalytic mechanism to that of SDH. Taken together, we suggest that KFR is a new member of the SDH family. Importance A limited number of species of acetic acid bacteria, such as Gluconobacter sp. strain CHM43, produce 5-ketofructose at a high yield, a potential low calorie sweetener. Here we show that an NADPH-dependent 5-ketofructose reductase (KFR) is involved in 5-ketofructose degradation and we characterize this enzyme with respect to its structure, phylogeny, and function. The crystal structure of KFR was similar to that of shikimate dehydrogenase, which is functionally crucial in the shikimate pathway in bacteria and plants. Phylogenetic analysis suggested that KFR is positioned in a small sub-group of the shikimate dehydrogenase family. Catalytically important amino acid residues were also conserved and their relevance was experimentally validated. Thus, we propose KFR as a new member of shikimate dehydrogenase family.

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