Conserved positively charged amino acid residues in the putative binding pocket are important for OATP1B1 function

Deoxyhypusine synthase catalyses the first step in the biosynthesis of hypusine [Nε-(4-amino-2-hydroxybutyl)lysine]. The crystal structure of human deoxyhypusine synthase in complex with NAD revealed four NAD-binding sites per enzyme tetramer, and led to a prediction of the spermidine-binding pocket. We have replaced each of the seven amino acid residues at the predicted spermidine-binding site, and eleven residues that contact NAD, on an individual basis with alanine. Of the amino acid residues at the spermidine site, substitution of Asp-243, Trp-327, His-288, Asp-316 or Glu-323 with alanine caused an almost complete loss of spermidine binding and enzyme activity; only the mutation Tyr-305 → Ala showed partial binding and activity. His-288 → Ala was also deficient in terms of binding NAD. NAD binding was significantly reduced in all of the NAD-site mutant enzymes, except for Glu-137 → Ala, which showed a normal binding of NAD, but was totally lacking in spermidine binding. Of the NAD-site mutant enzymes, Asp-342 → Ala, Asp-313 → Ala and Asp-238 → Ala displayed the lowest binding of NAD. These enzymes and His-288Ala also showed a reduced binding of spermidine, presumably because spermidine binding is dependent on NAD. These findings permit the positive identification of amino acid residues critical for binding of spermidine and NAD, and provide a new insight into the complex molecular interactions involved in the deoxyhypusine synthase reaction.

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Functional role of positively charged amino acid residues in the transmembrane segments of the yeast PMA1 ATPase

Folia Microbiologica ◽

10.1007/bf02816517 ◽

1998 ◽

Vol 43 (2) ◽

pp. 208-210 ◽

Cited By ~ 2

Author(s):

K. P. Padmanabha ◽

V. V. Petrov ◽

C. W. Slayman

Keyword(s):

Amino Acid ◽

Functional Role ◽

Amino Acid Residues ◽

Transmembrane Segments ◽

Positively Charged

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Determination of three amino acids causing alteration of proteolytic activities of staphylococcal glutamyl endopeptidases

Biological Chemistry ◽

10.1515/bc.2009.027 ◽

2009 ◽

Vol 390 (3) ◽

Cited By ~ 5

Author(s):

Takayuki K. Nemoto ◽

Toshio Ono ◽

Yu Shimoyama ◽

Shigenobu Kimura ◽

Yuko Ohara-Nemoto

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Protease Activity ◽

Binding Pocket ◽

Catalytic Triad ◽

Amino Acid Residues ◽

Proteolytic Activities ◽

Hydrophobic Amino Acids ◽

The Difference

Abstract Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus warneri secrete glutamyl endopeptidases, designated GluV8, GluSE, and GluSW, respectively. The order of their protease activities is GluSE<GluSW<<GluV8. In the present study, we investigated the mechanism that causes these differences. Expression of chimeric proteins between GluV8 and GluSE revealed that the difference is primarily attributed to amino acid residues 170–195, which define the intrinsic protease activity, and additionally to residues 119–169, which affect the proteolytic sensitivity. Among nine substitutions present in residues 170–195 of the three proteases, the substitutions at positions 185, 188, and 189 were responsible for the changes in their activities, and the combination of W185, V188, and P189, which naturally occurs in GluV8, exerts the highest protease activity. W185 and P189 were indispensable for full activity, but V188 could be replaced by hydrophobic amino acids. These three amino acid residues appear to create a substrate-binding pocket together with the catalytic triad and the N-terminal V1, and therefore define the K m values of the proteases. We also describe a method to produce a chimeric form of GluSE and GluV8 that is resistant to proteolysis, and therefore possesses 4-fold higher activity than the wild-type recombinant GluV8.

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Distribution of positively charged amino acid residues in antimicrobial peptide epinecidin-1 is crucial for in vitro glioblastoma cytotoxicity and its underlying mechanisms

Chemico-Biological Interactions ◽

10.1016/j.cbi.2019.108904 ◽

2020 ◽

Vol 315 ◽

pp. 108904 ◽

Cited By ~ 2

Author(s):

Bor-Chyuan Su ◽

Tsung-Han Wu ◽

Chun-Hua Hsu ◽

Jyh-Yih Chen

Keyword(s):

Amino Acid ◽

Antimicrobial Peptide ◽

Amino Acid Residues ◽

Underlying Mechanisms ◽

Positively Charged

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Investigations of Dimethylglycine, Glycine Betaine, and Ectoine Uptake by a Betaine-Carnitine-Choline Transporter Family Transporter with Diverse Substrate Specificity in Vibrio Species

Journal of Bacteriology ◽

10.1128/jb.00314-20 ◽

2020 ◽

Vol 202 (24) ◽

Author(s):

Gwendolyn J. Gregory ◽

Anirudha Dutta ◽

Vijay Parashar ◽

E. Fidelma Boyd

Keyword(s):

Amino Acid ◽

Glycine Betaine ◽

Vibrio Parahaemolyticus ◽

Compatible Solutes ◽

Binding Pocket ◽

Compatible Solute ◽

Amino Acid Residues ◽

Choline Transporter ◽

Content Type ◽

Highly Effective

ABSTRACT Fluctuations in osmolarity are one of the most prevalent stresses to which bacteria must adapt, both hypo- and hyperosmotic conditions. Most bacteria cope with high osmolarity by accumulating compatible solutes (osmolytes) in the cytoplasm to maintain the turgor pressure of the cell. Vibrio parahaemolyticus, a halophile, utilizes at least six compatible solute transporters for the uptake of osmolytes: two ABC family ProU transporters and four betaine-carnitine-choline transporter (BCCT) family transporters. The full range of compatible solutes transported by this species has yet to be determined. Using an osmolyte phenotypic microarray plate for growth analyses, we expanded the known osmolytes used by V. parahaemolyticus to include N,N-dimethylglycine (DMG), among others. Growth pattern analysis of four triple-bccT mutants, possessing only one functional BCCT, indicated that BccT1 (VP1456), BccT2 (VP1723), and BccT3 (VP1905) transported DMG. BccT1 was unusual in that it could take up both compounds with methylated head groups (glycine betaine [GB], choline, and DMG) and cyclic compounds (ectoine and proline). Bioinformatics analysis identified the four coordinating amino acid residues for GB in the BccT1 protein. In silico modeling analysis demonstrated that GB, DMG, and ectoine docked in the same binding pocket in BccT1. Using site-directed mutagenesis, we showed that a strain with all four residues mutated resulted in the loss of uptake of GB, DMG, and ectoine. We showed that three of the four residues were essential for ectoine uptake, whereas only one of the residues was important for GB uptake. Overall, we have demonstrated that DMG is a highly effective compatible solute for Vibrio species and have elucidated the amino acid residues in BccT1 that are important for the coordination of GB, DMG, and ectoine transport. IMPORTANCE Vibrio parahaemolyticus possesses at least six osmolyte transporters, which allow the bacterium to adapt to high-salinity conditions. In this study, we identified several additional osmolytes that were utilized by V. parahaemolyticus. We demonstrated that the compound DMG, which is present in the marine environment, was a highly effective osmolyte for Vibrio species. We determined that DMG is transported via BCCT family carriers, which have not been shown previously to take up this compound. BccT1 was a carrier for GB, DMG, and ectoine, and we identified the amino acid residues essential for the coordination of these compounds. The data suggest that for BccT1, GB is more easily accommodated than ectoine in the transporter binding pocket.

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Specific water-soluble substrates for chymotrypsin: Attempts for compensating diminished P1-S1 interactions

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19882884 ◽

1988 ◽

Vol 53 (11) ◽

pp. 2884-2889 ◽

Cited By ~ 9

Author(s):

Volker Schellenberger ◽

Ute Schellenberger ◽

Hans-Dieter Jakubke

Keyword(s):

Amino Acid ◽

Methyl Esters ◽

Water Soluble ◽

Amino Acid Residues ◽

Substrate Properties ◽

Ester Moiety ◽

Benzyl Esters ◽

Peptide Esters ◽

Acyl Donors ◽

Positively Charged

N-Maleyl-L-amino acid and peptide esters were synthesized and employed as substrates for α-chymotrypsin. From the kcat/KM values can be suggested that benzyl esters are significantly better substrates than the appropriate methyl esters. Further improvement in the substrate properties results from the introduction of the p-nitrobenzyl ester moiety. The choline ester of benzyloxycarbonyl-L-phenylalanine with the highest kcat/KM value confirmed the P1' leaving group specificity for positively charged residues. From the kinetic data can be concluded that acyl donors with high kcat/KM values, which are useful in kinetically controlled enzymatic peptide synthesis, need not contain aromatic amino acid residues in the P1 position.

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