J M Shaver | PubMed Health

PubMed1996

Single-amino acid substitutions eliminate lysine inhibition of maize dihydrodipicolinate synthase.

Dihydrodipicolinate synthase (DHPS; EC 4.2.1.52) catalyzes the first step in biosynthesis of lysine in plants and bacteria. DHPS in plants is highly sensitive to end-product inhibition by lysine and, therefore, has an important role in regulating metabolite flux into lysine. To better understand the feedback inhibition properties of the plant enzyme, we transformed a maize cDNA for lysine-sensitive DHPS into an Escherichia coli strain lacking DHPS activity. Cells were mutagenized with ethylmethanesulfonate, and potential DHPS mutants were selected by growth on minimal medium containing the inhibitory lysine analogue S-2-aminoethyl-L-cysteine. DHPS assays identified surviving colonies expressing lysine-insensitive DHPS activity. Ten single-base-pair mutations were identified in the maize DHPS cDNA sequence; these mutations were specific to one of three amino acid residues (amino acids 157, 162, and 166) localized within a short region of the polypeptide. No other mutations were present in the remaining DHPS cDNA sequence, indicating that altering only one of the three residues suffices to eliminate lysine inhibition of maize DHPS. Identification of these specific mutations that change the highly sensitive maize DHPS to a lysine-insensitive isoform will help resolve the lysine-binding mechanism and the resultant conformational changes involved in inhibition of DHPS activity. The plant-derived mutant DHPS genes may also be used to improve nutritional quality of maize or other cereal grains that have inadequate lysine content when fed to animals such as poultry, swine, or humans.

Amino Acid Sequence↗

PubMed1996

Maximum entropy method for frequency-domain fluorescence lifetime analysis. 2. Timing, mismatched intensity, and reference lifetime errors.

The maximum entropy method (MEM) provides a robust and unbiased solution to fluorescence lifetime data through the use of a broad window of decay terms fit by simultaneous minimization of the chi 2 goodness-of-fit parameter and maximization of a statistical entropy function. This work investigated the effects of three systematic errors, common in frequency-domain measurements, on fluorescence lifetime recovery by MEM. Through real and simulated data, the expression of the systematic errors in lifetime distributions recovered by MEM was compared to that in standard nonlinear least-squares (NLLS) analysis. Reference lifetime errors in the presence of random noise had similar effects on both MEM and NLLS results. Characteristic changes in the recovered lifetimes, fractional intensities, and peak shapes were related to the identification of the true reference lifetime. Compared to NLLS, MEM afforded significant improvements for the recovery of lifetimes and fractional intensities from data containing timing or mismatched intensity errors. These improvements are linked to the dynamic, self-modeling approach of MEM and the direction provided by the entropy criterion. These results speak to the utility of the maximum entropy approach in frequency-domain fluorescence lifetime recovery as well as in other applications.

Fluorescence↗