Roles Of Active Site Residues In Loda, A Cysteine Tryptophylquinone Dependent Ε-Lysine Oxidase

Keywords

Amine oxidase; Cofactor; Enzyme mechanism; Posttranslational modification; Quinoprotein; Redox enzyme

Abstract

Site-directed mutagenesis identified residues in the substrate channel of LodA that play multiple roles in regulating Km values of substrates, kcat and the extent of biosynthesis of the protein-derived cysteine tryptophylquinone (CTQ) cofactor. Mutations of Cys448 increase Km values for lysine and O2, with the larger effect on Klysine. Tyr211 resides within a mobile loop and is seen in the crystal structure of LodA to form a hydrogen bond with Lys530 that appears to stabilize its position in the channel. Y211F LodA had reduced levels of CTQ but near normal levels of kcat. K530A and K530R variants exhibited diminished levels of CTQ but significantly increased kcat. The Y211F, K530A and K530R mutations each caused large increases in the Km values for lysine and O2. These effects of the mutations of Tyr211 and Lys530 suggest that when these residues are hydrogen-bonded they may form a gate that controls entry and exit of substrates and products from the active site. Y211A and Y211E variants had the highest level of CTQ but exhibited no activity. These results highlight the different evolutionary factors that must be considered for enzymes which possess protein-derived cofactors, in which the catalytic cofactor must be generated by posttranslational modifications.

Publication Date

6-10-2015

Publication Title

Archives of Biochemistry and Biophysics

Volume

579

Number of Pages

26-32

Document Type

Article

Personal Identifier

scopus

DOI Link

https://doi.org/10.1016/j.abb.2015.05.013

Socpus ID

84930966169 (Scopus)

Source API URL

https://api.elsevier.com/content/abstract/scopus_id/84930966169

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