scholarly journals Improved selectivity of an engineered multi-product terpene synthase

2014 ◽  
Vol 12 (23) ◽  
pp. 4013-4020 ◽  
Author(s):  
Ryan Lauchli ◽  
Julia Pitzer ◽  
Rebekah Z. Kitto ◽  
Karolina Z. Kalbarczyk ◽  
Kersten S. Rabe
Keyword(s):  
Farnesyl Diphosphate ◽  
Terpene Synthase ◽  
Kinetic Properties ◽  
Natural Substrate ◽  
Wild Type ◽  
Random Mutation ◽  
Sesquiterpene Synthase ◽  
Germacrene D

Random mutation of the multi-product sesquiterpene synthase Cop2 generated a mutant that converted the natural substrate farnesyl diphosphate (FPP) into germacrene D-4-ol with 77% selectivity without detrimental effects on wild-type kinetic properties.

scholarly journals Gating Competence of Constitutively Open CLC-0 Mutants Revealed by the Interaction with a Small Organic Inhibitor

2003 ◽  
Vol 122 (3) ◽  
pp. 295-306 ◽  
Author(s):  
Sonia Traverso ◽  
Laura Elia ◽  
Michael Pusch
Keyword(s):  
Chloride Channels ◽  
Bound State ◽  
Side Chain ◽  
Pore Channel ◽  
Kinetic Properties ◽  
Wild Type ◽  
High Affinity ◽  
Critical Residue ◽  
Fast Gating

Opening of CLC chloride channels is coupled to the translocation of the permeant anion. From the recent structure determination of bacterial CLC proteins in the closed and open configuration, a glutamate residue was hypothesized to form part of the Cl−-sensitive gate. The negatively charged side-chain of the glutamate was suggested to occlude the permeation pathway in the closed state, while opening of a single protopore of the double-pore channel would reflect mainly a movement of this side-chain toward the extracellular pore vestibule, with little rearrangement of the rest of the channel. Here we show that mutating this critical residue (Glu166) in the prototype Torpedo CLC-0 to alanine, serine, or lysine leads to constitutively open channels, whereas a mutation to aspartate strongly slowed down opening. Furthermore, we investigated the interaction of the small organic channel blocker p-chlorophenoxy-acetic acid (CPA) with the mutants E166A and E166S. Both mutants were strongly inhibited by CPA at negative voltages with a >200-fold larger affinity than for wild-type CLC-0 (apparent KD at −140 mV ∼4 μM). A three-state linear model with an open state, a low-affinity and a high-affinity CPA-bound state can quantitatively describe steady-state and kinetic properties of the CPA block. The parameters of the model and additional mutagenesis suggest that the high-affinity CPA-bound state is similar to the closed configuration of the protopore gate of wild-type CLC-0. In the E166A mutant the glutamate side chain that occludes the permeation pathway is absent. Thus, if gating consists only in movement of this side-chain the mutant E166A should not be able to assume a closed conformation. It may thus be that fast gating in CLC-0 is more complex than anticipated from the bacterial structures.

scholarly journals The importance of the interdomain hinge in intramolecular electron transfer in flavocytochrome b2

1993 ◽  
Vol 291 (1) ◽  
pp. 89-94 ◽  
Author(s):  
P White ◽  
F D C Manson ◽  
C E Brunt ◽  
S K Chapman ◽  
G A Reid
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Cytochrome C ◽  
Kinetic Properties ◽  
Stopped Flow ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Cytochrome C Reductase ◽  
Flavocytochrome B2 ◽  
Cytochrome C Reduction

The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) are connected by a typical hinge peptide. The amino acid sequence of this interdomain hinge is dramatically different in flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala. This difference in the hinge is believed to contribute to the difference in kinetic properties between the two enzymes. To probe the importance of the hinge, an interspecies hybrid enzyme has been constructed comprising the bulk of the S. cerevisiae enzyme but containing the H. anomala flavocytochrome b2 hinge. The kinetic properties of this ‘hinge-swap’ enzyme have been investigated by steady-state and stopped-flow methods. The hinge-swap enzyme remains a good lactate dehydrogenase as is evident from steady-state experiments with ferricyanide as acceptor (only 3-fold less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (2.5-fold slower than in wild-type enzyme). The major effect of the hinge-swap mutation is to lower dramatically the enzyme's effectiveness as a cytochrome c reductase; kcat. for cytochrome c reduction falls by more than 100-fold, from 207 +/- 10 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 1.62 +/- 0.41 s-1 in the mutant enzyme. This fall in cytochrome c reductase activity results from poor interdomain electron transfer between the FMN and haem groups. This can be demonstrated by the fact that the kcat. for haem reduction in the hinge-swap enzyme (measured by the stopped-flow method) has a value of 1.61 +/- 0.42 s-1, identical with the value for cytochrome c reduction and some 300-fold lower than the value for the wild-type enzyme. From these and other kinetic parameters, including kinetic isotope effects with [2-2H]lactate, we conclude that the hinge plays a crucial role in allowing efficient electron transfer between the two domains of flavocytochrome b2.

scholarly journals Single Amino Acid Substitutions in HXT2.4 from Scheffersomyces stipitis Lead to Improved Cellobiose Fermentation by Engineered Saccharomyces cerevisiae

2012 ◽  
Vol 79 (5) ◽  
pp. 1500-1507 ◽  
Author(s):  
Suk-Jin Ha ◽  
Heejin Kim ◽  
Yuping Lin ◽  
Myoung-Uoon Jang ◽  
Jonathan M. Galazka ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Single Amino Acid ◽  
Kinetic Properties ◽  
Wild Type ◽  
Scheffersomyces Stipitis ◽  
Content Type ◽  
Charged Amino Acids ◽  
Cellodextrin Transporter ◽  
Engineered Strain

ABSTRACTSaccharomyces cerevisiaecannot utilize cellobiose, but this yeast can be engineered to ferment cellobiose by introducing both cellodextrin transporter (cdt-1) and intracellular β-glucosidase (gh1-1) genes fromNeurospora crassa. Here, we report that an engineeredS. cerevisiaestrain expressing the putative hexose transporter geneHXT2.4fromScheffersomyces stipitisandgh1-1can also ferment cellobiose. This result suggests that HXT2.4p may function as a cellobiose transporter whenHXT2.4is overexpressed inS. cerevisiae. However, cellobiose fermentation by the engineered strain expressingHXT2.4andgh1-1was much slower and less efficient than that by an engineered strain that initially expressedcdt-1andgh1-1. The rate of cellobiose fermentation by theHXT2.4-expressing strain increased drastically after serial subcultures on cellobiose. Sequencing and retransformation of the isolated plasmids from a single colony of the fast cellobiose-fermenting culture led to the identification of a mutation (A291D) in HXT2.4 that is responsible for improved cellobiose fermentation by the evolvedS. cerevisiaestrain. Substitutions for alanine (A291) of negatively charged amino acids (A291E and A291D) or positively charged amino acids (A291K and A291R) significantly improved cellobiose fermentation. The mutant HXT2.4(A291D) exhibited 1.5-fold higherKmand 4-fold higherVmaxvalues than those from wild-type HXT2.4, whereas the expression levels were the same. These results suggest that the kinetic properties of wild-type HXT2.4 expressed inS. cerevisiaeare suboptimal, and mutations of A291 into bulky charged amino acids might transform HXT2.4p into an efficient transporter, enabling rapid cellobiose fermentation by engineeredS. cerevisiaestrains.

scholarly journals Functional investigation of methanol dehydrogenase-like protein XoxF in Methylobacterium extorquens AM1

Microbiology ◽  
2010 ◽  
Vol 156 (8) ◽  
pp. 2575-2586 ◽  
Author(s):  
Sabrina Schmidt ◽  
Philipp Christen ◽  
Patrick Kiefer ◽  
Julia A. Vorholt
Keyword(s):  
Growth Parameters ◽  
Methanol Dehydrogenase ◽  
Plant Colonization ◽  
Kinetic Properties ◽  
Wild Type ◽  
Methylobacterium Extorquens ◽  
Experimental Conditions ◽  
Dependent Protein ◽  
One Carbon Metabolism ◽  
Functional Investigation

Methanol dehydrogenase-like protein XoxF of Methylobacterium extorquens AM1 exhibits a sequence identity of 50 % to the catalytic subunit MxaF of periplasmic methanol dehydrogenase in the same organism. The latter has been characterized in detail, identified as a pyrroloquinoline quinone (PQQ)-dependent protein, and shown to be essential for growth in the presence of methanol in this methylotrophic model bacterium. In contrast, the function of XoxF in M. extorquens AM1 has not yet been elucidated, and a phenotype remained to be described for a xoxF mutant. Here, we found that a xoxF mutant is less competitive than the wild-type during colonization of the phyllosphere of Arabidopsis thaliana, indicating a function for XoxF during plant colonization. A comparison of the growth parameters of the M. extorquens AM1 xoxF mutant with those of the wild-type during exponential growth revealed a reduced methanol uptake rate and a reduced growth rate for the xoxF mutant of about 30 %. Experiments with cells starved for carbon revealed that methanol oxidation in the xoxF mutant occurs less rapidly compared with the wild-type, especially in the first minutes after methanol addition. A distinct phenotype for the xoxF mutant was also observed when formate and CO2 production were measured after the addition of methanol or formaldehyde to starved cells. The wild-type, but not the xoxF mutant, accumulated formate upon substrate addition and had a 1 h lag in CO2 production under the experimental conditions. Determination of the kinetic properties of the purified enzyme showed a conversion capacity for both formaldehyde and methanol. The results suggest that XoxF is involved in one-carbon metabolism in M. extorquens AM1.

cAMP-dependent protein kinase regulates renal epithelial cell properties

1991 ◽  
Vol 260 (6) ◽  
pp. C1290-C1299 ◽  
Author(s):  
K. Amsler ◽  
S. Ghatani ◽  
B. A. Hemmings
Keyword(s):  
Protein Kinase ◽  
Epithelial Cell ◽  
Cell Line ◽  
Cell Junctions ◽  
Kinetic Properties ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Renal Epithelial Cell ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

Previous studies have implicated adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) in regulation of both growth and expression of differentiated function in the pig renal epithelial cell, LLC-PK1. To investigate this possible regulatory mechanism, we compared growth behavior, morphology, and appearance of two differentiated functions, Na-hexose symport (SYMP) and gamma-glutamyl transpeptidase (gamma-GT), in the LLC-PK1 line and two PKA-deficient mutants (FIB4 and FIB6). Compared with the wild-type cell line, the mutant lines continued to proliferate at higher population densities and exhibited altered cell morphology, poorer formation of the brush-border structure, and decreased or lack of expression of SYMP and gamma-GT activities. Wild-type and mutant cells exhibit an identical logarithmic growth rate. Both lines form cell-cell junctions and exhibit identical kinetic properties of expressed SYMP activity. These results strongly support the hypothesis that PKA modulates a defined subset of cellular processes, including aspects of growth control and expression of the differentiated phenotype, in this renal epithelial cell line.

scholarly journals Inhibition of Inositol Phosphorylceramide Synthase by the Cyclic Peptide Aureobasidin A

2009 ◽  
Vol 53 (2) ◽  
pp. 496-504 ◽  
Author(s):  
Paul A. Aeed ◽  
Casey L. Young ◽  
Marek M. Nagiec ◽  
Åke P. Elhammer
Keyword(s):  
Cyclic Peptide ◽  
Time Dependent ◽  
Mutant Enzyme ◽  
Kinetic Properties ◽  
Dependent Manner ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Noncompetitive Inhibitor ◽  
Aureobasidin A ◽  
Comparison Of The Results

ABSTRACT By using a detergent-washed membrane preparation, the interaction of the fungal natural product inhibitor aureobasidin A (AbA) with inositol phosphorylceramide synthase (IPC synthase) was studied by kinetic analysis of wild-type and mutant enzyme-catalyzed reactions. AbA inhibited the wild-type enzyme from both Candida albicans and Saccharomyces cerevisiae in an irreversible, time-dependent manner, with apparent Ki values of 183 and 234 pM, respectively. Three synthetic chemistry-derived AbA derivatives, PHA-533179, PHA-556655, and PHA-556656, had affinities 4 to 5 orders of magnitude lower and were reversible inhibitors that competed with the donor substrate phosphatidylinositol (PI). AbA was a reversible, apparently noncompetitive inhibitor, with a Ki of 1.4 μM, of the IPC synthase from an AbA-resistant S. cerevisiae mutant. The Km values for both substrates (ceramide and PI) were similar when they interacted with the mutant and the wild-type enzymes. By contrast, the V max for the mutant enzyme was less than 10% of that for the wild-type enzyme. A comparison of the results obtained with AbA with those obtained with two other natural products inhibitors, rustmicin and khafrefungin, revealed that while rustmicin appeared to be a reversible, noncompetitive inhibitor of the wild-type enzyme, with a Ki of 16.0 nM, khafrefungin had the kinetic properties of a time-dependent inhibitor and an apparent Ki of 0.43 nM. An evaluation of the efficiencies of these compounds as inhibitors of the mutant enzyme revealed for both a drop in the apparent affinity for the enzyme of more than 2 orders of magnitude.

scholarly journals Dissociation of the tetrameric phosphoglycerate mutase from yeast by a mutation in the subunit contact region

1993 ◽  
Vol 295 (3) ◽  
pp. 743-748 ◽  
Author(s):  
M F White ◽  
L A Fothergill-Gilmore ◽  
S M Kelly ◽  
N C Price
Keyword(s):  
Contact Region ◽  
Chloride Concentration ◽  
Mutant Enzyme ◽  
Phosphoglycerate Mutase ◽  
Kinetic Properties ◽  
Wild Type ◽  
Guanidinium Chloride ◽  
Wild Type Enzyme ◽  
Tetrameric Structure ◽  
Subunit Contact

Phosphoglycerate mutases from different sources exhibit a variety of quaternary structures (tetramer, dimer and monomer). To perturb the tetrameric structure of yeast phosphoglycerate mutase we have prepared a mutant enzyme in which Lys-168 in the subunit-contact region has been replaced by proline. The K168P mutant enzyme undergoes dissociation to dimers at low concentrations; thus on lowering the concentration from 200 micrograms/ml to 5 micrograms/ml the proportion of tetramer falls from 85% to 53%. The tetrameric structure of the wild-type enzyme remains intact over this range of concentrations. The mutant enzyme has similar kinetic properties to the wild-type enzyme, with kcat. being reduced by 26%. Far-u.v. c.d. studies show that there has been a small loss of helical structure in the mutant. Compared with wild-type enzyme, the K168P mutant enzyme is slightly less stable towards proteolysis by trypsin, but significantly less stable towards denaturation by guanidinium chloride, with the midpoint concentration of guanidinium chloride some 50% lower. After denaturation, the mutant enzyme could regain activity and quaternary structure when the guanidinium chloride concentration was lowered to 0.05 M. The properties of the mutant enzyme are discussed in terms of other dimeric phosphoglycerate and bisphosphoglycerate mutases which contain proline at position 168.

Kinetic properties of cathepsin D and BACE 1 indicate the need to search for additional β-secretase candidate(s)

2008 ◽  
Vol 389 (3) ◽  
pp. 313-320 ◽  
Author(s):  
Israel Schechter ◽  
Etty Ziv
Keyword(s):  
Cathepsin D ◽  
Kinetic Properties ◽  
Wild Type ◽  
Western Blots ◽  
Knock Out ◽  
Worldwide Population ◽  
Secretase Activity ◽  
Brain Extracts ◽  
Pepstatin A ◽  
Bace 1

Abstract Many studies suggest that BACE 1 is the genuine β-secretase; however, this is not undisputed. The wild-type (WT) β-site of the amyloid precursor protein (APP) present in the worldwide population is cleaved very slowly (k cat/K m: approx. 50 m -1 s-1), while proteases acting on relevant substrates are much more efficient (k cat/K m: 104–106 m -1 s-1). Knock-out of BACE 1 in mouse markedly reduces Aβ formation. Nevertheless, studies in other systems show that knock-out experiments in rodents and corresponding genetic defects in human may reveal different phenotypes. Considering these issues, we searched for other β-secretase candidate(s), identified cathepsin D, and evaluated properties of cathepsin D related to BACE 1 that were not examined previously. The kinetic constants (k cat, K m, k cat/K m) for cleaving peptides with β-sites of the WT or the mutated Swedish families (SW) APP by human BACE 1 and cathepsin D were determined and found to be similar. Western blots reveal that in human brain cathepsin D is approximately 280-fold more abundant than BACE 1. Furthermore, pepstatin A strongly inhibits the cleavage of SW and WT peptides by both brain extracts and cathepsin D, but not by BACE 1. These findings indicate that β-secretase activity observed in brain extracts is mainly due to cathepsin D. Nevertheless, as both BACE 1 and cathepsin D show poor activity towards the WT β-site sequence, it is necessary to continue the search for additional β-secretase candidate(s).

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