Functional Mutants of Yeast Alcohol Dehydrogenase

Wills, Christopher; Kratofil, Paul; Martin, Tracy

doi:10.1007/978-1-4684-4142-0_24

Cited by 8 publications

(16 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Many, but not all, of the mutated ADHs from resistant strains had altered electrophoretic mobilities consistent with additional positive charge, such as the H44R, W54R, W82R and P316R enzymes [4]. Our results with the H15R enzyme (not selected from a genetic screen) show that just increasing the positive charge of the ADH does not necessarily produce a fitter yeast.…”

Section: Discussionmentioning

confidence: 78%

“…On the other hand, the kinetic constants for the W54R enzyme differed by 5-fold or more (Table 1 cf. [4]), but the kinetic constants for this enzyme were difficult to determine because they were so large. The W54R substitution is in the active site (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…In a pioneering study of evolution of fitter organisms in the laboratory, Wills and coworkers selected for yeast strains that must grow by fermenting glucose while being exposed to allyl alcohol [1-4]. The yeast require alcohol dehydrogenase I (ADH1, cytoplasmic isoenzyme 1) to reduce acetaldehyde for anaerobic growth (NADH + acetaldehyde == NAD + + ethanol), but oxidation of allyl alcohol (CH 2 =CH-CH 2 OH) with NAD + to acrolein (CH 2 =CH-CHO) catalyzed by ADH is lethal to wild-type yeast.…”

Section: Introductionmentioning

confidence: 99%

“…Decreased ADH activity can be expected to cause slower growth, due to slower reduction of acetaldehyde, and higher NADH/NAD + levels, which should decrease the rate of oxidation of allyl alcohol and be protective. Changes in redox state in mutant yeasts were observed, but it has not been clear what changes in kinetic constants for the substituted ADHs could account for the altered redox state and the increased fitness [4-6]. …”

Section: Introductionmentioning

confidence: 99%

“…Amino acid substitutions were identified by protein sequencing methods in four different ADH1s to be H44R, W54R, W82 (to an unidentified basic amino acid), and P316R [4,7]. We previously produced the H44R enzyme by site-directed mutagenesis and concluded that 5-fold decreases in turnover numbers and catalytic efficiencies could explain the growth of fitter yeast [8].…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Bradykinetic alcohol dehydrogenases make yeast fitter for growth in the presence of allyl alcohol

Plapp

Lee

Khanna

et al. 2013

Chemico-Biological Interactions

View full text Add to dashboard Cite

Previous studies showed that fitter yeast (Saccharomyces cerevisiae) that can grow by fermenting glucose in the presence of allyl alcohol, which is oxidized by alcohol dehydrogenase I (ADH1) to toxic acrolein, had mutations in the ADH1 gene that led to decreased ADH activity. These yeast may grow more slowly due to slower reduction of acetaldehyde and a higher NADH/NAD+ ratio, which should decrease the oxidation of allyl alcohol. We determined steady-state kinetic constants for three yeast ADHs with new site-directed substitutions and examined the correlation between catalytic efficiency and growth on selective media of yeast expressing six different ADHs. The H15R substitution (a test for electrostatic effects) is on the surface of ADH and has small effects on the kinetics. The H44R substitution (affecting interactions with the coenzyme pyrophosphate) was previously shown to decrease affinity for coenzymes 2-4-fold and turnover numbers (V/Et) by 4-6-fold. The W82R substitution is distant from the active site, but decreases turnover numbers by 5-6-fold, perhaps by effects on protein dynamics. The E67Q substitution near the catalytic zinc was shown previously to increase the Michaelis constant for acetaldehyde and to decrease turnover for ethanol oxidation. The W54R substitution, in the substrate binding site, increases kinetic constants (K’s, by > 10-fold) while decreasing turnover numbers by 2-7-fold. Growth of yeast expressing the different ADHs on YPD plates (yeast extract, peptone and dextrose) plus antimycin to require fermentation, was positively correlated with the log of catalytic efficiency for the sequential bi reaction (V1/KiaKb = KeqV2/KpKiq, varying over 4 orders of magnitude, adjusted for different levels of ADH expression) in the order: WT H15R > H44R > W82R > E67Q > W54R. Growth on YPD plus 10 mM allyl alcohol was inversely correlated with catalytic efficiency. The fitter yeast are “bradytrophs” (slow growing) because the ADHs have decreased catalytic efficiency.

show abstract

Section: Discussionmentioning

confidence: 78%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Bradykinetic alcohol dehydrogenases make yeast fitter for growth in the presence of allyl alcohol

Plapp

Lee

Khanna

et al. 2013

Chemico-Biological Interactions

View full text Add to dashboard Cite

show abstract

Exploring the relationship between genotype and phenotype using yeast alcohol dehydrogenase 1

Krzysiak

Doyle

Huff

2021

Biochem Molecular Bio Educ

View full text Add to dashboard Cite

Project-based (research-driven) laboratory courses stimulate student involvement, improve critical thinking and initiate cooperative learning. To this end, a 7-week laboratory project was designed for a sophomore cell biology course to reinforce the fundamental relationship between genotype and phenotype using yeast alcohol dehydrogenase I (ADH1). Working in pairs, students used site-directed mutagenesis to create a H44R mutation in the ADH1 gene sequence inserted into a YEp13 shuttle vector. These plasmids were propagated in E. coli, sequenced, and reintroduced into a yeast strain expressing no ADH1 activity. The growth patterns on selective media were determined. As the mutation allows for growth in the presence of allyl alcohol, students make the connection between DNA sequence and protein function. Student performance was assessed with pre-and post-tests, with improvement observed across all learning objectives. In addition to meeting the learning outcomes, 98% of the students thought that this experience allowed them to see how the scientific process can encompass multiple techniques to answer a single question. Eighty-four percent of the students thought that this experience was more engaging than other lab experiences they have had. Our multi-week laboratory examining the phenotypic changes in yeast alcohol metabolism successfully developed students' understanding of the scientific process, knowledge of molecular techniques and the relationship of gene sequence to protein function in an engaging manner.

show abstract

Structural Evolution of Yeast Alcohol Dehydrogenase in the Laboratory

Wills

1984

Microorganisms as Model Systems for Studying Evolution

View full text Add to dashboard Cite

Functional Mutants of Yeast Alcohol Dehydrogenase

Cited by 8 publications

References 28 publications

Bradykinetic alcohol dehydrogenases make yeast fitter for growth in the presence of allyl alcohol

Bradykinetic alcohol dehydrogenases make yeast fitter for growth in the presence of allyl alcohol

Exploring the relationship between genotype and phenotype using yeast alcohol dehydrogenase 1

Structural Evolution of Yeast Alcohol Dehydrogenase in the Laboratory

Contact Info

Product

Resources

About