Dataset Information

Reliable Metabolic Flux Estimation in Escherichia coli Central Carbon Metabolism Using Intracellular Free Amino Acids.

ABSTRACT: 13C metabolic flux analysis (MFA) is a tool of metabolic engineering for investigation of in vivo flux distribution. A direct 13C enrichment analysis of intracellular free amino acids (FAAs) is expected to reduce time for labeling experiments of the MFA. Measurable FAAs should, however, vary among the MFA experiments since the pool sizes of intracellular free metabolites depend on cellular metabolic conditions. In this study, minimal 13C enrichment data of FAAs was investigated to perform the FAAs-based MFA. An examination of a continuous culture of Escherichia coli using 13C-labeled glucose showed that the time required to reach an isotopically steady state for FAAs is rather faster than that for conventional method using proteinogenic amino acids (PAAs). Considering 95% confidence intervals, it was found that the metabolic flux distribution estimated using FAAs has a similar reliability to that of the PAAs-based method. The comparative analysis identified glutamate, aspartate, alanine and phenylalanine as the common amino acids observed in E. coli under different culture conditions. The results of MFA also demonstrated that the 13C enrichment data of the four amino acids is required for a reliable analysis of the flux distribution.

SUBMITTER: Okahashi N

PROVIDER: S-EPMC4101513 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Reliable Metabolic Flux Estimation in Escherichia coli Central Carbon Metabolism Using Intracellular Free Amino Acids.

Okahashi Nobuyuki N Kajihata Shuichi S Furusawa Chikara C Shimizu Hiroshi H

Metabolites 20140530 2

13C metabolic flux analysis (MFA) is a tool of metabolic engineering for investigation of in vivo flux distribution. A direct 13C enrichment analysis of intracellular free amino acids (FAAs) is expected to reduce time for labeling experiments of the MFA. Measurable FAAs should, however, vary among the MFA experiments since the pool sizes of intracellular free metabolites depend on cellular metabolic conditions. In this study, minimal 13C enrichment data of FAAs was investigated to perform the FA ...[more]

PMID: 24957033

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Project description:The E. coli UvrD protein is a nonhexameric DNA helicase that belongs to superfamily I and plays a crucial role in both nucleotide excision repair and methyl-directed mismatch repair. Previous data suggested that wild-type UvrD has optimal activity in its oligomeric form. However, crystal structures of the UvrD-DNA complex were only resolved for monomeric UvrD, using a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C). However, biochemical findings performed using UvrDΔ40C indicated that this mutant failed to dimerize, although its DNA-unwinding activity was comparable to that of wild-type UvrD. Although the C-terminus plays essential roles in nucleic acid binding for many proteins with helicase and dimerization activities, the exact function of the C-terminus is poorly understood. Thus, to understand the function of the C-terminal amino acids of UvrD, we performed single-molecule direct visualization. Photobleaching of dye-labeled UvrDΔ40C molecules revealed that two or three UvrDΔ40C molecules could bind simultaneously to an 18-bp double-stranded DNA with a 20-nucleotide, 3' single-stranded DNA tail in the absence of ATP. Simultaneous visualization of association/dissociation of the mutant with/from DNA and the DNA-unwinding dynamics of the mutant in the presence of ATP demonstrated that, as with wild-type UvrD, two or three UvrDΔ40C molecules were primarily responsible for DNA unwinding. The determined association/dissociation rate constants for the second bound monomer were ∼2.5-fold larger than that of wild-type UvrD. The involvement of multiple UvrDΔ40C molecules in DNA unwinding was also observed under a physiological salt concentration (200 mM NaCl). These results suggest that multiple UvrDΔ40C molecules, which may form an oligomer, play an active role in DNA unwinding in vivo and that deleting the C-terminal 40 residues altered the interaction of the second UvrD monomer with DNA without affecting the interaction with the first bound UvrD monomer.

Dataset Information

Reliable Metabolic Flux Estimation in Escherichia coli Central Carbon Metabolism Using Intracellular Free Amino Acids.

Publications

Reliable Metabolic Flux Estimation in Escherichia coli Central Carbon Metabolism Using Intracellular Free Amino Acids.

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