Dataset Information

Combine Approach HNE - Combined proteomic approaches for the identification of specific amino acid residues modified by 4-hydroxy-2-nonenal under physiological conditions

ABSTRACT: HNE modifications

INSTRUMENT(S): instrument model, LTQ

SUBMITTER: MÂªLuisa hernÃ¡ez

PROVIDER: PRD000286 | Pride | 2010-10-21

REPOSITORIES: pride

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Dataset's files

Source:

			Action	DRS
	PRIDE_Exp_Complete_Ac_13513.pride.mgf.gz	Mgf
	PRIDE_Exp_Complete_Ac_13513.xml.gz	Xml
	PRIDE_Exp_Complete_Ac_13514.pride.mgf.gz	Mgf
	PRIDE_Exp_Complete_Ac_13514.xml.gz	Xml

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Publications

Combined proteomic approaches for the identification of specific amino acid residues modified by 4-hydroxy-2-nonenal under physiological conditions.

Mendez Dario D Hernaez Maria Luisa ML Diez Amalia A Puyet Antonio A Bautista Jose M JM

Journal of proteome research 20101008 11

Proteins modified by 4-hydroxy-2-nonenal (HNE) are cellular markers of oxidative stress in health and disease. HNE is generated by free radical chain reactions during oxidative stress as a major end-product of the oxidative fatty acid metabolism. Identification and quantitative analysis of HNE-modified proteins are readily performed by using specific antibodies raised against them. Further on, the identification of the amino acid residues involved in the HNE-modification is an additional step in ...[more]

PMID: 20818828

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Project description:The enzyme AHAS (acetohydroxy acid synthase), which is involved in the biosynthesis of valine, leucine and isoleucine, is the target of several classes of herbicides. A model of tobacco AHAS was generated based on the X-ray structure of yeast AHAS. Well conserved residues at the herbicide-binding site were identified, and the roles of three of these residues (Phe-205, Val-570 and Phe-577) were determined by site-directed mutagenesis. The Phe-205 mutants F205A, F205H, F205W and F205Y showed markedly decreased levels of catalytic efficiency, and cross-resistance to two or three classes of herbicides, i.e. Londax (a sulphonylurea herbicide), Cadre (an imidazolinone herbicide) and TP (a triazolopyrimidine derivative). None of the mutations caused significant changes in the secondary or tertiary structure of the enzyme. Four mutants of Phe-577, i.e. F577D, F577E, F577K and F577R, showed unaltered V(max) values, but substantially decreased catalytic efficiency. However, these mutants were highly resistant to two or three of the tested herbicides. The three mutants F577D, F577E and F577R had a similar secondary structure to that of wild-type AHAS. Conservative mutations of Phe-577, i.e. F577W and F577Y, did not affect the kinetic properties of the enzyme or its inhibition by herbicides. The mutation Val-570 to Asn abolished the binding affinity of the enzyme for FAD as well as its activity, and also caused a change in the tertiary structure of AHAS. However, the mutant V570Q was active, but resistant to two classes of herbicides, i.e. Londax and TP. The conservative mutant V570I was substantially reduced in catalytic efficiency and moderately resistant to the three herbicides. The results of this study suggest that residues Phe-205, Val-570 and Phe-577 in tobacco AHAS are located at or near the binding site that is common for the three classes of herbicides. In addition, Phe-205 and Val-570 are probably located at the herbicide-binding site that may overlap partially with the active site. Selected mutants of Phe-577 are expected to be utilized to construct herbicide-resistant transgenic plants.