Covalent flavinylation of L-aspartate oxidase from Escherichia coli using N6-(6-carboxyhexyl)-FAD succinimidoester.

Negri, A; Buckmann, A F; Tedeschi, G; Stocker, Achim; Ceciliani, F; Treu, C; Ronchi, S (1999). Covalent flavinylation of L-aspartate oxidase from Escherichia coli using N6-(6-carboxyhexyl)-FAD succinimidoester. Journal of protein chemistry, 18(6), pp. 671-676. Springer 10.1023/a:1020606323716

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L-Aspartate oxidase is a flavoprotein catalyzing the first step in the de novo biosynthesis of pyridine nucleotides in E. coli. Binding of FAD to L-aspartate oxidase is relatively weak (K(d) 6.7 x 10(-7) M), resulting in partial loss of the coenzyme under many experimental conditions. Only the three-dimensional structure of the apo-enzyme has been obtained so far. In order to probe the flavin-binding site of the enzyme, apo-L-aspartate oxidase has been reacted with N6-(6-carboxyhexyl)-FAD succinimidoester. The structural characterization of the resulting N6-(6-carbamoylxyhexyl)FAD-L-aspartate oxidase shows the covalent incorporation of 1 FAD-analog/monomer. Residue Lys38 was identified as the target of the covalent modification. N6-(6-carbamoylxyhexyl)-FAD-L-aspartate oxidase shows only 2% catalytic activity as compared to the native enzyme. Comparison of some properties of the flavinylated and native enzymes suggests that, although the flavin is covalently bound to the former in the region predicted from molecular modeling studies, the microenvironment around the isoallossazine is different in the two forms.

Item Type:

Journal Article (Review Article)

Division/Institute:

08 Faculty of Science > Department of Chemistry, Biochemistry and Pharmaceutical Sciences (DCBP)

UniBE Contributor:

Stocker, Achim

Subjects:

500 Science > 570 Life sciences; biology
500 Science > 540 Chemistry

ISSN:

0277-8033

Publisher:

Springer

Language:

English

Submitter:

Anja Ebeling

Date Deposited:

11 Mar 2020 13:32

Last Modified:

21 Apr 2021 15:12

Publisher DOI:

10.1023/a:1020606323716

PubMed ID:

10609643

BORIS DOI:

10.7892/boris.141763

URI:

https://boris.unibe.ch/id/eprint/141763

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