LodB is required for the recombinant synthesis of the quinoprotein l-lysine-ε-oxidase from Marinomonas mediterranea

Abstract

Marinomonas mediterranea is a marine gamma-proteobacterium that synthesizes LodA, a novel L-lysine-ε-oxidase (E.C. 1.4.3.20). This enzyme oxidizes L-lysine generating 2-aminoadipate 6-semialdehyde, ammonium, and hydrogen peroxide. Unlike other L-amino acid oxidases, LodA is not a flavoprotein but contains a quinone cofactor. LodA is encoded by an operon with two genes, lodA and lodB. In the native system, LodB is required for the synthesis of a functional LodA. In this study, we report the recombinant expression of LodA in Escherichia coli using vectors that allow its expression and accumulation in the cytoplasm. To reveal the L-lysine-ε-oxidase activity using the Amplex Red method for hydrogen peroxide detection, it is necessary to first remove the E. coli cytoplasmic catalases. The flavoprotein LodB is the only M. mediterranea protein required in the recombinant system for the generation of the cofactor of LodA. In the absence of LodB, LodA does not contain the quinone cofactor and remains in an inactive form. The results presented indicate that LodB participates in the posttranslational modification of LodA that generates the quinone cofactor.

Roles of active site residues in LodA, a cysteine tryptophylquinone dependent ε-lysine oxidase

“…This is beneficial because causing death of a subpopulation of cells facilitates biofilm differentiation and dispersal of surviving cells with phenotypic variation among dispersed cells [4]. Wild type (WT) LodA has been recombinantly expressed in E. coli [5] and isolated. LodA contains a protein-derived cofactor [6] which is generated by posttranslational modifications.…”

“…The cofactor is cysteine tryptophylquinone (CTQ) which is formed by the di-oxygenation of Trp581 and the crosslinking of this residue with Cys516 (Figure 1). Expression of active LodA with correctly synthesized CTQ requires the co-expression of the lodB gene [5], which encodes a flavoprotein that is required for the posttranslational modifications that generate CTQ.…”

Heterologous production of l-lysine ε-oxidase by directed evolution using a fusion reporter method

“…15) Here, we detected LodA activity in the cells of E. coli BL21 (DE3), and Chacón-Verdú et al also expressed the activity of LodA intracellularly in E. coli Rosetta (DE3) pRARE. 2) These results indicate that M. mediterranea has a unique LodA transport system in the wild-type strain. T646T(ACT-ACG), and the mutant enzyme was named LodA7mut (Fig.…”

“…1(B)) of LodA. 2,3) However, the mechanism of the biosynthesis of the quinone cofactor by LodB is not elucidated. Recently, our group applied LodA for quantification of L-lysine, 4) but it could not be purified in high yields, owing to melanin pigments synthesized by tyrosinase (EC 1.14.18.1) in M. mediterranea.…”

“…6) Recently, Chacón-Verdú et al reported that LodA was not expressed in Escherichia coli BL21 (DE3), but was expressed intracellularly in E. coli Rosetta (DE3) pRARE by cultivation at low temperature. 2) LodA was expressed by the translation of the additional tRNAs provided by pRARE in the expression system. But in their expression system, the E. coli cytoplasmic catalases must be removed to detect LodA activity.…”