Faculty Opinions recommendation of Key amino acid residues conferring enhanced enzyme activity at cold temperatures in an Antarctic polyextremophilic β-galactosidase.

2017 ◽  
Author(s):  
Qiang Cui
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Amino Acid Residues ◽  
Cold Temperatures

scholarly journals Structure–function studies of human deoxyhypusine synthase: identification of amino acid residues critical for the binding of spermidine and NAD

2001 ◽  
Vol 355 (3) ◽  
pp. 841-849 ◽  
Author(s):  
Chang Hoon LEE ◽  
Patrick Y. UM ◽  
Myung Hee PARK
Keyword(s):  
Crystal Structure ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Binding Sites ◽  
Binding Pocket ◽  
Complete Loss ◽  
Amino Acid Residues ◽  
Deoxyhypusine Synthase ◽  
Positive Identification ◽  
Insight Into

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.

scholarly journals Biosynthesis in vitro of tryptophanase by polyribosomes from induced cultures of Escherichia coli

1971 ◽  
Vol 123 (3) ◽  
pp. 355-365 ◽  
Author(s):  
S. A. M. Khairul Bashar ◽  
J. H. Parish ◽  
Marjorie Brown
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Enzyme Synthesis ◽  
Supernatant Fraction ◽  
Amino Acid Residues ◽  
Radioactive Amino Acid ◽  
Tryptophanase Activity

1. Polyribosomes were isolated from Escherichia coli grown in media in which tryptophanase is induced and in which it is repressed. The polyribosomes from the induced bacteria had a small amount of tryptophanase activity associated with them. 2. A portion of the enzyme activity remained bound to polyribosomes during centrifuging in sucrose gradients. 3. Incubation of tryptophanase-containing polyribosomes with puromycin released enzyme activity. 4. The binding of the enzyme to the polyribosomes did not depend on the presence of DNA. 5. When the polyribosomes were incubated under conditions of protein synthesis with supernatant fraction obtained from repressed bacteria, a small but statistically significant increase in enzyme activity was produced. 6. When a radioactive amino acid was included in the incubation mixture for the tryptophanase system a radioactive protein was obtained whose chromatographic, electrophoretic and sedimentation properties were identical with those of tryptophanase. 7. The amount of incorporation was consistent with the amount of new enzyme synthesis predicted by the increase in enzyme activity. Both radioactive incorporation and increase in enzyme activity were shown to be energy-dependent and also negative controls were obtained by using zero-time incubations or polyribosomes isolated from either repressed cells or a mutant lacking the ability to produce tryptophanase. 8. The distribution of radioactive leucine in the carboxyl region of the newly labelled tryptophanase was examined by digesting the labelled protein with carboxypeptidases. It was shown that the radioactivity was more highly concentrated towards the carboxyl terminus when the incubation times for protein synthesis were shorter (implying that, with longer incubation times, longer lengths of polypeptide chain contained radioactive amino acid residues).

scholarly journals Evidence for essential histidine and dicarboxylic amino-acid residues in the active site of UDP-glucose : solasodine glucosyltransferase from eggplant leaves.

2003 ◽  
Vol 50 (2) ◽  
pp. 567-572 ◽  
Author(s):  
Paulina Nawłoka ◽  
Małgorzata Kalinowska ◽  
Cezary Paczkowski ◽  
Zdzisław A Wojciechowski
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Aspartic Acid ◽  
Active Site ◽  
Inhibitory Effect ◽  
Amino Acid Residues ◽  
Chemical Probes ◽  
Dicarboxylic Amino Acid ◽  
Enzyme Substrates ◽  
Arginine Residues

Effects of several chemical probes selectively modifying various amino-acid residues on the activity of UDP-glucose : solasodine glucosyltransferase from eggplant leaves was studied. It was shown that diethylpyrocarbonate (DEPC), a specific modifier of histidine residues, was strongly inhibitory. However, in the presence of excessive amounts of the enzyme substrates, i.e. either UDP-glucose or solasodine, the inhibitory effect of DEPC was much weaker indicating that histidine (or histidines) are present in the active site of the enzyme. Our results suggest also that unmodified residues of glutamic (or aspartic) acid, lysine, cysteine, tyrosine and tryptophan are necessary for full activity of the enzyme. Reagents modifying serine and arginine residues have no effect on the enzyme activity.

scholarly journals Variant Amino Acid Residues Alter the Enzyme Activity of Peanut Type 2 Diacylglycerol Acyltransferases

2017 ◽  
Vol 8 ◽  
Author(s):  
Ling Zheng ◽  
Jay Shockey ◽  
Fei Bian ◽  
Gao Chen ◽  
Lei Shan ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Amino Acid Residues ◽  
Variant Amino Acid

scholarly journals Separate bisphosphatase domain of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: the role of the C-terminal tail in modulating enzyme activity

1997 ◽  
Vol 328 (3) ◽  
pp. 751-756 ◽  
Author(s):  
Lin LI ◽  
Song LING ◽  
Chun-lai WU ◽  
Wei-zhe YAO ◽  
Gen-jun XU
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Substrate Inhibition ◽  
Fold Increase ◽  
Chicken Liver ◽  
Bifunctional Enzyme ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Km Value ◽  
The Difference

The separate bisphosphatase domain (amino acid residues 243-468) of the chicken liver bifunctional enzyme 6-phosphofructo-2-kinase-fructose-2,6-bisphosphatase was expressed in Escherichia coli and purified to homogeneity. The fructose-2,6-bisphosphatase activity of the separate domain was 7-fold higher than that of the native bifunctional enzyme, and exhibited substrate inhibition characteristic of the native enzyme. The inhibition of the enzymes by fructose 2,6-bisphosphate could be overcome by Pi, glycerol 3-phosphate and GTP. Deletion of 30 amino acid residues from the C-terminus of the separate domain resulted in around a 5-fold increase in the Vmax of the bisphosphatase. Also, the truncated form was more accessible to chemical modification by diethyl pyrocarbonate and N-ethylmaleimide, suggesting a more open structure than the wild-type form. In addition, the mutation of cysteine-389 to alanine increased bisphosphatase activity by 20% and the Km value for fructose 2,6-bisphosphate by 3-fold, and both the point mutation at cysteine-389 and the deletional mutation led to the predominantly insoluble expression of the enzyme. The results indicated that the C-terminal tail plays a role in modulating the enzyme activity and suggested that the difference in the C-terminal tail sequence is responsible for the difference in activity of the chicken and rat liver fructose-2,6-bisphosphatases. It is postulated that an interaction between the C-terminal tail and the active site might be present.

scholarly journals Contribution of Phe-7 to Tat-Dependent Export of β-Lactamase in Xanthomonas campestris

2012 ◽  
Vol 56 (7) ◽  
pp. 3597-3602 ◽  
Author(s):  
Chen-Wei Lee ◽  
Yi-Hsuan Tseng ◽  
Fu-Seng Deng ◽  
Juey-Wen Lin ◽  
Yi-Hsiung Tseng ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Protein Structure ◽  
Amino Acid ◽  
Xanthomonas Campestris ◽  
Constitutive Expression ◽  
Amino Acid Substitutions ◽  
Amino Acid Residues ◽  
Content Type ◽  
Aromatic Interactions ◽  
Tat Pathway

ABSTRACTStrains ofXanthomonas campestrispv. campestris isolated in Taiwan are commonly resistant to ampicillin owing to the constitutive expression of a chromosomally encoded β-lactamase that is secreted into the periplasm. In this study, we found that levels of β-lactamase vary amongX. campestrispv. campestris strains, a difference that can be attributed to amino acid substitutions at least at positions 7 and 206, with the former having the major impact. Bioinformatic and PCR analyses indicated thatX. campestrispv. campestris possessestatABCgenes and that the signal peptide ofX. campestrispv. campestris pre-Bla contains the typical twin-arginine motif (N-R-R-Q-F-L at amino acid residues 3 to 8 in strainX. campestrispv. campestris strain 11), suggesting that Bla is secreted via the Tat pathway. To assess the importance of Phe7in the efficient export ofX. campestrispv. campestris Bla, we prepared mutant constructs containing amino acid substitutions and monitored their expression by measuring enzyme activity and detecting Bla protein by Western blotting. The results indicate that replacement of Phe7with Leu severely inhibited Bla export whereas replacement with Pro almost abolished it. Although a change to Arg caused moderate inhibition of export, replacement with Tyr had no effect. These results suggest that for efficient export of Bla byX. campestrispv. campestris, the aromatic-aromatic interactions and stability of protein structure around the twin-arginine motif are important, since only proteins that can attain a folded state in the cytoplasm are competent for export via the Tat pathway.

scholarly journals Key amino acid residues conferring enhanced enzyme activity at cold temperatures in an Antarctic polyextremophilic β-galactosidase

2017 ◽  
Vol 114 (47) ◽  
pp. 12530-12535 ◽  
Author(s):  
Victoria J. Laye ◽  
Ram Karan ◽  
Jong-Myoung Kim ◽  
Wolf T. Pecher ◽  
Priya DasSarma ◽  
...  
Keyword(s):  
Amino Acid ◽  
Comparative Genomics ◽  
Active Site ◽  
Interdisciplinary Approach ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Temperature Dependent ◽  
Temperature Function ◽  
Cold Temperatures

The Antarctic microorganism Halorubrum lacusprofundi harbors a model polyextremophilic β-galactosidase that functions in cold, hypersaline conditions. Six amino acid residues potentially important for cold activity were identified by comparative genomics and substituted with evolutionarily conserved residues (N251D, A263S, I299L, F387L, I476V, and V482L) in closely related homologs from mesophilic haloarchaea. Using a homology model, four residues (N251, A263, I299, and F387) were located in the TIM barrel around the active site in domain A, and two residues (I476 and V482) were within coiled or β-sheet regions in domain B distant to the active site. Site-directed mutagenesis was performed by partial gene synthesis, and enzymes were overproduced from the cold-inducible cspD2 promoter in the genetically tractable Haloarchaeon, Halobacterium sp. NRC-1. Purified enzymes were characterized by steady-state kinetic analysis at temperatures from 0 to 25 °C using the chromogenic substrate o-nitrophenyl-β-galactoside. All substitutions resulted in altered temperature activity profiles compared with wild type, with five of the six clearly exhibiting reduced catalytic efficiency (kcat/Km) at colder temperatures and/or higher efficiency at warmer temperatures. These results could be accounted for by temperature-dependent changes in both Km and kcat (three substitutions) or either Km or kcat (one substitution each). The effects were correlated with perturbation of charge, hydrogen bonding, or packing, likely affecting the temperature-dependent flexibility and function of the enzyme. Our interdisciplinary approach, incorporating comparative genomics, mutagenesis, enzyme kinetics, and modeling, has shown that divergence of a very small number of amino acid residues can account for the cold temperature function of a polyextremophilic enzyme.

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