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Electrochemical probing of in vivo 5-hydroxymethyl furfural reduction in Saccharomyces cerevisiae.
Anal Chem. 2009 Dec 15; 81(24):9896-901.AC

Abstract

In this work, mediated amperometry was used to evaluate whether differences in intracellular nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) level could be observed between a genetically modified Saccharomyces cerevisiae strain, engineered for NADPH dependent 5-hydroxymethyl-2-furaldehyde (HMF) reduction, and its control strain. Cells overexpressing the alcohol dehydrogenase 6 gene (ADH6 strain) and cells carrying the corresponding control plasmid (control strain) were each immobilized on Au-microelectrodes. The real-time dynamics of NAD(P)H availability in the two strains, preincubated with HMF, was probed using the menadione-ferricyanide double mediator system. A lower intracellular NADPH level as the consequence of more effective HMF reduction was observed for the ADH6 strain both with and without added glucose, which increases the overall cellular NADPH level. The mediated amperometric signal during real-time monitoring of the concentration dependent HMF reduction in living cells could be translated into the cellular enzyme kinetic parameters: K(M,cell)(app), V(MAX), k(cat,cell), and k(cat,cell)/K)M,cell)(app). The results indicated that the overexpression of the ADH6 gene gave a 68% decrease in K(M,cell)(app) and 42% increase in V(MAX), resulting in a 4-fold increase in k(cat,cell)/K(M,cell)(app). These results demonstrate that the mediated amperometric method is useful for monitoring the short-term dynamics of NAD(P)H variations and determining cellular enzyme kinetic parameters in S. cerevisiae cells.

Authors+Show Affiliations

Department of Applied Microbiology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

19925001

Citation

Kostesha, Natalie V., et al. "Electrochemical Probing of in Vivo 5-hydroxymethyl Furfural Reduction in Saccharomyces Cerevisiae." Analytical Chemistry, vol. 81, no. 24, 2009, pp. 9896-901.
Kostesha NV, Almeida JR, Heiskanen AR, et al. Electrochemical probing of in vivo 5-hydroxymethyl furfural reduction in Saccharomyces cerevisiae. Anal Chem. 2009;81(24):9896-901.
Kostesha, N. V., Almeida, J. R., Heiskanen, A. R., Gorwa-Grauslund, M. F., Hahn-Hägerdal, B., & Emnéus, J. (2009). Electrochemical probing of in vivo 5-hydroxymethyl furfural reduction in Saccharomyces cerevisiae. Analytical Chemistry, 81(24), 9896-901. https://doi.org/10.1021/ac901402m
Kostesha NV, et al. Electrochemical Probing of in Vivo 5-hydroxymethyl Furfural Reduction in Saccharomyces Cerevisiae. Anal Chem. 2009 Dec 15;81(24):9896-901. PubMed PMID: 19925001.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Electrochemical probing of in vivo 5-hydroxymethyl furfural reduction in Saccharomyces cerevisiae. AU - Kostesha,Natalie V, AU - Almeida,João R M, AU - Heiskanen,Arto R, AU - Gorwa-Grauslund,Marie F, AU - Hahn-Hägerdal,Barbel, AU - Emnéus,Jenny, PY - 2009/11/21/entrez PY - 2009/11/21/pubmed PY - 2010/2/25/medline SP - 9896 EP - 901 JF - Analytical chemistry JO - Anal Chem VL - 81 IS - 24 N2 - In this work, mediated amperometry was used to evaluate whether differences in intracellular nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) level could be observed between a genetically modified Saccharomyces cerevisiae strain, engineered for NADPH dependent 5-hydroxymethyl-2-furaldehyde (HMF) reduction, and its control strain. Cells overexpressing the alcohol dehydrogenase 6 gene (ADH6 strain) and cells carrying the corresponding control plasmid (control strain) were each immobilized on Au-microelectrodes. The real-time dynamics of NAD(P)H availability in the two strains, preincubated with HMF, was probed using the menadione-ferricyanide double mediator system. A lower intracellular NADPH level as the consequence of more effective HMF reduction was observed for the ADH6 strain both with and without added glucose, which increases the overall cellular NADPH level. The mediated amperometric signal during real-time monitoring of the concentration dependent HMF reduction in living cells could be translated into the cellular enzyme kinetic parameters: K(M,cell)(app), V(MAX), k(cat,cell), and k(cat,cell)/K)M,cell)(app). The results indicated that the overexpression of the ADH6 gene gave a 68% decrease in K(M,cell)(app) and 42% increase in V(MAX), resulting in a 4-fold increase in k(cat,cell)/K(M,cell)(app). These results demonstrate that the mediated amperometric method is useful for monitoring the short-term dynamics of NAD(P)H variations and determining cellular enzyme kinetic parameters in S. cerevisiae cells. SN - 1520-6882 UR - https://www.unboundmedicine.com/medline/citation/19925001/Electrochemical_probing_of_in_vivo_5_hydroxymethyl_furfural_reduction_in_Saccharomyces_cerevisiae_ L2 - https://doi.org/10.1021/ac901402m DB - PRIME DP - Unbound Medicine ER -