scholarly journals Engineering Candida tenuis Xylose Reductase for Improved Utilization of NADH: Antagonistic Effects of Multiple Side Chain Replacements and Performance of Site-Directed Mutants under Simulated In Vivo Conditions

2005 ◽  
Vol 71 (10) ◽  
pp. 6390-6393 ◽  
Author(s):  
Barbara Petschacher ◽  
Bernd Nidetzky
Keyword(s):  
Xylose Reductase ◽  
Binding Pocket ◽  
Kinetic Mechanism ◽  
Side Chain ◽  
Multiple Site ◽  
Wild Type ◽  
Structural Effects ◽  
Phosphate Binding ◽  
And Performance

ABSTRACT Six single- and multiple-site variants of Candida tenuis xylose reductase that were engineered to have side chain replacements in the coenzyme 2′-phosphate binding pocket were tested for NADPH versus NADH selectivity (R sel) in the presence of physiological reactant concentrations. The experimental R sel values agreed well with predictions from a kinetic mechanism describing mixed alternative coenzyme utilization. The Lys-274→Arg and Arg-280→His substitutions, which individually improved wild-type R sel 50- and 20-fold, respectively, had opposing structural effects when they were combined in a double mutant.

scholarly journals Mutant tryptophan aporepressors with altered specificities of corepressor recognition.

Genetics ◽  
1991 ◽  
Vol 128 (1) ◽  
pp. 29-35
Author(s):  
D N Arvidson ◽  
M Shapiro ◽  
P Youderian
Keyword(s):  
Dna Binding ◽  
Binding Pocket ◽  
Side Chain ◽  
Wild Type ◽  
Mutant Proteins ◽  
Naturally Occurring ◽  
Repressor Complex ◽  
Genes Encoding ◽  
Active Repressor

Abstract The Escherichia coli trpR gene encodes tryptophan aporepressor, which binds the corepressor ligand, L-tryptophan, to form an active repressor complex. The side chain of residue valine 58 of Trp aporepressor sits at the bottom of the corepressor (L-tryptophan) binding pocket. Mutant trpR genes encoding changes of Val58 to the other 19 naturally occurring amino acids were made. Each of the mutant proteins requires a higher intracellular concentration of tryptophan for activation of DNA binding than wild-type aporepressor. Whereas wild-type aporepressor is activated better by 5-methyltryptophan (5-MT) than by tryptophan, Ile58 and other mutant aporepressors prefer tryptophan to 5-MT as corepressor, and Ala58 and Gly58 prefer 5-MT much more strongly than wild-type aporepressor in vivo. These mutant aporepressors are the first examples of DNA-binding proteins with altered specificities of cofactor recognition.

A study of the relationships of interactions between Asp-201, Na+ or K+, and galactosyl C6 hydroxyl and their effects on binding and reactivity of β-galactosidase

2004 ◽  
Vol 82 (2) ◽  
pp. 275-284 ◽  
Author(s):  
Julia Xu ◽  
Mary A.A McRae ◽  
Scott Harron ◽  
Beatrice Rob ◽  
Reuben E Huber
Keyword(s):  
Physical Properties ◽  
Negative Charge ◽  
Binding Pocket ◽  
Side Chain ◽  
Wild Type ◽  
Sodium Potassium ◽  
The Difference ◽  
Potassium Binding ◽  
Covalent Intermediate ◽  
Catalytic Effects

The interactions between Na+ (and K+) and Asp-201 of β-galactosidase were studied. Analysis of the changes in Km and Vmax showed that the Kd for Na+ of wild type β-galactosidase (0.36 ± 0.09 mM) was about 10× lower than for K+ (3.9 ± 0.6 mM). The difference is probably because of the size and other physical properties of the ions and the binding pocket. Decreases of Km as functions of Na+ and K+ for oNPG and pNPG and decreases of the Ki of both shallow and deep mode inhibitors were similar, whereas the Km and Ki of substrates and inhibitors without C6 hydroxyls remained constant. Thus, Na+ and K+ are important for binding galactosyl moieties via the C6 hydroxyl throughout catalysis. Na+ and K+ had lesser effects on the Vmax. The Vmax of pNPF and pNPA (substrates that lack a C6 hydroxyl) did not change upon addition of Na+ or K+, showing that the catalytic effects are also mediated via the C6 hydroxyl. Arrhenius plots indicated that Na+, but not K+, caused k3 (degalactosylation) to increase. Na+ also caused the k2 (galactosylation) with oNPG, but not with pNPG, to increase. In contrast, K+ caused the k2 values with both oNPG and pNPG to increase. Na+ and K+ mainly altered the entropies of activation of k2 and k3 with only small effects on the enthalpies of activation. This strongly suggests that only the positioning of the substrate, transition states, and covalent intermediate are altered by Na+ and K+. Further evidence that positioning is important was that substitution of Asp-201 with a Glu caused the Km and Ki values to increase significantly. In addition, the Kd values for Na+ or K+ were 5 to 8 fold higher. The negative charge of Asp-201 was shown to be vital for Na+ and K+ binding. Large amounts of Na+ or K+ had no effect on the very large Km and Ki values of D201N-β-galactosidase and the Vmax values changed minimally and in a linear rather than hyperbolic way. D201F-β-galactosidase, with a very bulky hydrophobic side chain in place of Asp, essentially obliterated all binding and catalysis.Key words: β-galactosidase, sodium, potassium, binding, aspartic acid.

Mutational analysis of the glucagon receptor: similarities with the vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide (PACAP)/secretin receptors for recognition of the ligand's third residue

2002 ◽  
Vol 362 (2) ◽  
pp. 389-394 ◽  
Author(s):  
Jason PERRET ◽  
Mélanie VAN CRAENENBROECK ◽  
Ingrid LANGER ◽  
Pascale VERTONGEN ◽  
Françoise GREGOIRE ◽  
...  
Keyword(s):  
Vasoactive Intestinal Peptide ◽  
Functional Properties ◽  
Mutational Analysis ◽  
Transmembrane Helix ◽  
Binding Pocket ◽  
Side Chain ◽  
Wild Type ◽  
Glucagon Receptor ◽  
Pituitary Adenylate Cyclase ◽  
In The Wild

Receptor recognition by the Asp3 residues of vasoactive intestinal peptide and secretin requires the presence of a lysine residue close to the second transmembrane helix (TM2)/first extracellular loop junction and an ionic bond with an arginine residue in TM2. We tested whether the glucagon Gln3 residue recognizes the equivalent positions in its receptor. Our data revealed that the binding and functional properties of the wild-type glucagon receptor and the K188R mutant were not significantly different, whereas all agonists had markedly lower potencies and affinities at the I195K mutated receptor. In contrast, glucagon was less potent and the Asp3-, Asn3- and Glu3-glucagon mutants were more potent and efficient at the double-mutated K188R/I195K receptor. Furthermore, these alterations were selective for position 3 of glucagon, as shown by the functional properties of the mutant Glu9- and Lys15-glucagon. Our results suggest that although the Gln3 residue of glucagon did not interact with the equivalent binding pocket as the Asp3 residue of vasoactive intestinal peptide or secretin, the Asp3-glucagon analogue was able to interact with position 188 of the K188R/I195K glucagon receptor. Nevertheless, the Gln3 side chain of glucagon probably binds very close to this region in the wild-type receptor.

scholarly journals Structural heterogeneity in biliverdin modulates spectral properties of Sandercyanin fluorescent protein

2021 ◽  
Author(s):  
Swagatha Ghosh ◽  
Sayan Mondal ◽  
Keerti Yadav ◽  
Shantanu Aggarwal ◽  
Wayne F. Schaefer ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Spectral Properties ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Binding Pocket ◽  
Structural Heterogeneity ◽  
Wild Type ◽  
Absorbing States

Sandercyanin, a blue homo-tetrameric lipocalin protein purified from Canadian walleye (Stizostedion vitreus), is the first far-red fluorescent protein reported in vertebrates. Sandercyanin binds non-covalently to biliverdin IXα (BLA) and fluoresces at 675nm on excitation at 375nm and 635nm. Sandercyanin fluorescence can be harnessed for many in vivo applications when engineered into a stable monomeric form. Here, we report the spectral properties and crystal structures of engineered monomeric Sandercyanin-BLA complexes. Compared to wild-type protein, monomeric Sandercyanin (~18kDa) binds BLA with similar affinities and show a broad red- shifted absorbance spectra but possess reduced quantum efficiency. Crystal structures reveal D-ring pyrrole of BLA rotated around the C14-C15 bond, which is stabilized by neighboring aromatic residues and increased water-mediated polar contacts in the BLA-binding pocket. A tetrameric Sandercyanin variant (Tyr-142-Ala) co-displaying red- and far-red absorbing states, and reduced fluorescence shows similar conformational changes in BLA binding pocket. Our results suggest that D-ring flexibility of BLA and its rearrangement reduces the fluorescence quantum-yield of monomeric Sandercyanin. Structures of monomeric Sandercyanin could be utilized as prototypes to generate bright BLA-inducible fluorescent proteins. Further, our study postulates a mechanism for modulating photo-states in BLA- bound lipocalins, known only in phytochromes till date.

scholarly journals Protection of cisplatin cytotoxicity by an inactive cyclin-dependent kinase

2010 ◽  
Vol 299 (1) ◽  
pp. F112-F120 ◽  
Author(s):  
Rawad Hodeify ◽  
Judit Megyesi ◽  
Adel Tarcsafalvi ◽  
Robert L. Safirstein ◽  
Peter M. Price
Keyword(s):  
Cell Death ◽  
Cyclin A ◽  
Binding Pocket ◽  
Cyclin Dependent Kinase ◽  
Wild Type ◽  
Cytochrome C Release ◽  
Endonuclease G ◽  
Cdk2 Activity

Cisplatin cytotoxicity is dependent on cyclin-dependent kinase 2 (Cdk2) activity in vivo and in vitro. A Cdk2 mutant (Cdk2-F80G) was designed in which the ATP-binding pocket was altered. When expressed in mouse kidney cells, this protein was kinase inactive, did not inhibit endogenous Cdk2, but protected from cisplatin. The mutant was localized in the cytoplasm, but when coexpressed with cyclin A, it was activated, localized to the nucleus, and no longer protected from cisplatin cytotoxicity. Cells exposed to cisplatin in the presence of the activated mutant had an apoptotic phenotype, and endonuclease G was released from mitochondria similar to that mediated by endogenous Cdk2. But unlike apoptosis mediated by wild-type Cdk2, cisplatin exposure of cells expressing the activated mutant did not cause cytochrome c release or significant caspase-3 activation. We conclude that cisplatin likely activates both caspase-dependent and -independent cell death, and Cdk2 is required for both pathways. The mutant-inactive Cdk2 protected from both death pathways, but after activation by excess cyclin A, caspase-independent cell death predominated.

scholarly journals Phosphate-binding pocket in Dicer-2 PAZ domain for high-fidelity siRNA production

2016 ◽  
Vol 113 (49) ◽  
pp. 14031-14036 ◽  
Author(s):  
Suresh K. Kandasamy ◽  
Ryuya Fukunaga
Keyword(s):  
Rna Silencing ◽  
Point Mutations ◽  
Binding Pocket ◽  
Rna Cleavage ◽  
High Fidelity ◽  
Small Interfering Rnas ◽  
Phosphate Binding ◽  
Micro Rnas ◽  
Paz Domain

The enzyme Dicer produces small silencing RNAs such as micro-RNAs (miRNAs) and small interfering RNAs (siRNAs). In Drosophila, Dicer-1 produces ∼22–24-nt miRNAs from pre-miRNAs, whereas Dicer-2 makes 21-nt siRNAs from long double-stranded RNAs (dsRNAs). How Dicer-2 precisely makes 21-nt siRNAs with a remarkably high fidelity is unknown. Here we report that recognition of the 5′-monophosphate of a long dsRNA substrate by a phosphate-binding pocket in the Dicer-2 PAZ (Piwi, Argonaute, and Zwille/Pinhead) domain is crucial for the length fidelity, but not the efficiency, in 21-nt siRNA production. Loss of the length fidelity, meaning increased length heterogeneity of siRNAs, caused by point mutations in the phosphate-binding pocket of the Dicer-2 PAZ domain decreased RNA silencing activity in vivo, showing the importance of the high fidelity to make 21-nt siRNAs. We propose that the 5′-monophosphate of a long dsRNA substrate is anchored by the phosphate-binding pocket in the Dicer-2 PAZ domain and the distance between the pocket and the RNA cleavage active site in the RNaseIII domain corresponds to the 21-nt pitch in the A-form duplex of a long dsRNA substrate, resulting in high-fidelity 21-nt siRNA production. This study sheds light on the molecular mechanism by which Dicer-2 produces 21-nt siRNAs with a remarkably high fidelity for efficient RNA silencing.

scholarly journals A novel SH2 recognition mechanism recruits Spt6 to the doubly phosphorylated RNA polymerase II linker at sites of transcription

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Matthew A Sdano ◽  
James M Fulcher ◽  
Sowmiya Palani ◽  
Mahesh B Chandrasekharan ◽  
Timothy J Parnell ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Sh2 Domain ◽  
Heptad Repeat ◽  
Side Chain ◽  
Phosphate Binding ◽  
Recognition Mechanism ◽  
Repressive Chromatin ◽  
Repeat Domain

We determined that the tandem SH2 domain of S. cerevisiae Spt6 binds the linker region of the RNA polymerase II subunit Rpb1 rather than the expected sites in its heptad repeat domain. The 4 nM binding affinity requires phosphorylation at Rpb1 S1493 and either T1471 or Y1473. Crystal structures showed that pT1471 binds the canonical SH2 pY site while pS1493 binds an unanticipated pocket 70 Å distant. Remarkably, the pT1471 phosphate occupies the phosphate-binding site of a canonical pY complex, while Y1473 occupies the position of a canonical pY side chain, with the combination of pT and Y mimicking a pY moiety. Biochemical data and modeling indicate that pY1473 can form an equivalent interaction, and we find that pT1471/pS1493 and pY1473/pS1493 combinations occur in vivo. ChIP-seq and genetic analyses demonstrate the importance of these interactions for recruitment of Spt6 to sites of transcription and for the maintenance of repressive chromatin.

scholarly journals Electrostatic stabilization in a pre-organized polar active site: the catalytic role of Lys-80 in Candida tenuis xylose reductase (AKR2B5) probed by site-directed mutagenesis and functional complementation studies

2005 ◽  
Vol 389 (2) ◽  
pp. 507-515 ◽  
Author(s):  
Regina Kratzer ◽  
Bernd Nidetzky
Keyword(s):  
Carbonyl Group ◽  
Acid Catalysis ◽  
Xylose Reductase ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Linear Free Energy Relationship ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
General Acid ◽  
Group Reduction

Lys-80 of Candida tenuis xylose reductase (AKR2B5) is conserved throughout the aldo–keto reductase protein superfamily and may prime the nearby Tyr-51 for general acid catalysis to NAD(P)H-dependent carbonyl group reduction. We have examined the catalytic significance of side-chain substitutions in two AKR2B5 mutants, Lys-80→Ala (K80A) and Asp-46→Asn Lys-80→Ala (D46N K80A), using steady-state kinetic analysis and restoration of activity with external amines. Binding of NAD+ (Kd=24 μM) and NADP+ (Kd=0.03 μM) was 10- and 40-fold tighter in K80A than the wild-type enzyme, whereas binding of NADH (Kd=51 μM) and NADPH (Kd=19 μM) was weakened 2- and 16-fold in this mutant respectively. D46N K80A bound NAD(P)H and NAD(P)+ uniformly approx. 5-fold less tightly than the wild-type enzyme. The second-order rate constant for non-covalent restoration of NADH-dependent reductase activity (kmax/Kamine) by protonated ethylamine was 0.11 M−1·s−1 for K80A, whereas no detectable rescue occurred for D46N K80A. After correction for effects of side-chain hydrophobicity, we obtained a linear free energy relationship of log (kmax/Kamine) and amine group pKa (slope=+0.29; r2=0.93) at pH 7.0. pH profiles of log (kcat/Km) for carbonyl group reduction by wild-type and D46N K80A revealed identical and kinetically unperturbed pKa values of 8.50 (±0.20). Therefore the protonated side chain of Lys-80 is not an essential activator of general acid catalysis by AKR2B5. Stabilized structurally through the salt-link interaction with the negatively charged Asp-46, it is proposed to pull the side chain of Tyr-51 into the catalytic position, leading to a preorganized polar environment of overall neutral charge, in which approximation of uncharged reactive groups is favoured and thus hydride transfer from NAD(P)H is strongly preferred. Lys-80 affects further the directional preference of AKR2B5 for NAD(P)H-dependent reduction by increasing NAD(P)H compared with NAD(P)+-binding selectivity.

scholarly journals Introduction of a Carboxyl Group in the First Transmembrane Helix of Escherichia coliF1Fo ATPase Subunit c and Cytoplasmic pH Regulation

2001 ◽  
Vol 183 (5) ◽  
pp. 1524-1530 ◽  
Author(s):  
Phil C. Jones
Keyword(s):  
Ph Regulation ◽  
Transmembrane Helix ◽  
Binding Pocket ◽  
Side Chain ◽  
Ion Binding ◽  
Cytoplasmic Ph ◽  
Wild Type ◽  
Atpase Subunit ◽  
Subunit C ◽  
E Coli

ABSTRACT The multicopy subunit c of the H+-transporting F1Fo ATP synthase of Escherichia coli folds across the membrane as a hairpin of two hydrophobic α helices. The subunits interact in a front-to-back fashion, forming an oligomeric ring with helix 1 packing in the interior and helix 2 at the periphery. A conserved carboxyl, Asp61 in E. coli, centered in the second transmembrane helix is essential for H+ transport. A second carboxylic acid in the first transmembrane helix is found at a position equivalent to Ile28 in several bacteria, some the cause of serious infectious disease. This side chain has been predicted to pack proximal to the essential carboxyl in helix 2. It appears that in some of these bacteria the primary function of the enzyme is H+pumping for cytoplasmic pH regulation. In this study, Ile28was changed to Asp and Glu. Both mutants were functional. However, unlike the wild type, the mutants showed pH-dependent ATPase-coupled H+ pumping and passive H+ transport through Fo. The results indicate that the presence of a second carboxylate enables regulation of enzyme function in response to cytoplasmic pH and that the ion binding pocket is aqueous accessible. The presence of a single carboxyl at position 28, in mutants I28D/D61G and I28E/D61G, did not support growth on a succinate carbon source. However, I28E/D61G was functional in ATPase-coupled H+transport. This result indicates that the side chain at position 28 is part of the ion binding pocket.

scholarly journals Conserved residues in Ycf54 are required for protochlorophyllide formation in Synechocystis sp. PCC 6803

2017 ◽  
Vol 474 (5) ◽  
pp. 667-681 ◽  
Author(s):  
Sarah Hollingshead ◽  
Sophie Bliss ◽  
Patrick J. Baker ◽  
C. Neil Hunter
Keyword(s):  
Hydrogen Bonding ◽  
Protoporphyrin Ix ◽  
Wild Type ◽  
Structural Effects ◽  
Conserved Residues ◽  
Functional Differences ◽  
In Vivo Effects ◽  
Mgpme Cyclase ◽  
Synechocystis Sp

Chlorophylls (Chls) are modified tetrapyrrole molecules, essential for photosynthesis. These pigments possess an isocyclic E ring formed by the Mg-protoporphyrin IX monomethylester cyclase (MgPME–cyclase). We assessed the in vivo effects of altering seven highly conserved residues within Ycf54, which is required for MgPME–cyclase activity in the cyanobacterium Synechocystis. Synechocystis strains harbouring the Ycf54 alterations D39A, F40A and R82A were blocked to varying degrees at the MgPME–cyclase step, whereas the A9G mutation reduced Ycf54 levels by ∼75%. Wild-type (WT) levels of the cyclase subunit CycI are present in strains with D39A and F40A, but these strains have lowered cellular Chl and photosystem accumulation. CycI is reduced by ∼50% in A9G and R82A, but A9G has no perturbations in Chl or photosystem accumulation, whilst R82A contains very little Chl and few photosystems. When FLAG tagged and used as bait in pulldown experiments, the three mutants D39A, F40A and R82A were unable to interact with the MgPME–cyclase component CycI, whereas A9G pulled down a similar level of CycI as WT Ycf54. These observations suggest that a stable interaction between CycI and Ycf54 is required for unimpeded Pchlide biosynthesis. Crystal structures of the WT, A9G and R82A Ycf54 proteins were solved and analysed to investigate the structural effects of these mutations. A loss of the local hydrogen bonding network and a reversal in the surface charge surrounding residue R82 are probably responsible for the functional differences observed in the R82A mutation. We conclude that the Ycf54 protein must form a stable interaction with CycI to promote optimal Pchlide biosynthesis.

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