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

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.

Interaction of GoxA with Its Modifying Enzyme and Its Subunit Assembly Are Dependent on the Extent of Cysteine Tryptophylquinone Biosynthesis

“…In contrast to what was observed with GoxA, no complex of LodA or LodA precursors with LodB was observed during purification. Interestingly, the preparation of pure LodA was found to contain a significant fraction of a precursor that lacked CTQ but no LodB 18 . Thus, while preLodA and LodB must also interact to achieve CTQ biosynthesis, the complex formation between those two proteins must be transient as was the case with preMADH and MauG, and different from what was observed for precursor GoxA-GoxB complex.…”

Roles of Conserved Residues of the Glycine Oxidase GoxA in Controlling Activity, Cooperativity, Subunit Composition, and Cysteine Tryptophylquinone Biosynthesis

“…This was verified by the crystal structure of LodA with the lysine-CTQ adduct formed (9). The roles of residues in and around the active site of the LodA in catalysis and CTQ biogenesis were identified by site-directed mutagenesis (10).…”

“…Phe-237 is of interest because it corresponds in the primary sequence to Tyr-211 of LodA (Fig. 2), which is conserved in most Group I proteins and which strongly influenced the K m values of both lysine and O 2 for LodA (10). In the homology model, the position of Phe-237 is not structurally conserved with Tyr-211 in LodA, as are the positions of CTQ, Asp-547/Asp-512 and His-466/Cys-448 (GoxA/LodA numbering, respectively).…”

“…His-466 -His-466 corresponds to the Cys-448 of LodA. This LodA residue was previously mutated to Ala, Asp, and His (10). No stable C448H LodA protein was isolated, suggesting that the mutation affected the biosynthesis or stability of the protein, or both.…”

Roles of Copper and a Conserved Aspartic Acid in the Autocatalytic Hydroxylation of a Specific Tryptophan Residue during Cysteine Tryptophylquinone Biogenesis

“…Also, it is shown that the total soluble D512A LodA exhibits no significant specificity for binding copper over other metals. It was previously shown that mutagenesis of other residues, such as Cys448, in the active-site and substrate channel of LodA effect the levels of mature CTQ present in the enzyme as well as kinetic parameters for the catalytic reaction 42 . Thus, perturbations of the positions of other residues in the active site which are caused by the D512A mutation or binding of a metal other than copper could also abolish the initial hydroxylation.…”