Catalytic mechanism of ancestral L-lysine oxidase assigned by sequence data mining

Abstract

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Reaction Mechanism of Ancestral l-Lys α-Oxidase from Caulobacter Species Studied by Biochemical, Structural, and Computational Analysis

“…The concentration of l -Lys in the samples with (filled circle) or without plasma (filled square) was estimated by an end-point assay at different concentrations of l -Lys, indicating that AncLLysO2 can apply to quantify l -Lys under conditions containing 0–400 μM l -Lys. Slopes on the plots had almost identical values to each other (Figure D) as well as to other l -amino acid quantification enzymes. ,, …”

“…The mixture was centrifuged, and LC-HRMS analysis was performed. The reaction scheme of AncLLysO2 was proposed by referring to the reaction mechanism of L-LOX/MOG and AncLLysO. − The peak of l -Lys completely disappeared after the addition of AncLLysO2, suggesting that AncLLysO2 reacts with l -Lys as expected (b and c for l -Lys in Figure ). At the same time, after consumption of l -Lys by AncLLysO2, reaction products were identified: 5-APNA, 5-APNM, and 3,4,5,6-tetrahydropyradine-2-carboxylic acid (TPCA) (b and c in Figure ).…”

“…Each of the enzymes generates distinct compounds as main products. 5-Amino pentanamide (5-APNM) and 5-amino pentanoic acid (5-APNA) are mainly produced by L-LOX/MOG and AncLLysO, respectively. , Because of this background, main products of AncLLysO2 may be different from L-LOX/MOG and AncLLysO.…”

“…There was a difference between AncLLysO and AncLLysO2 in the production of 5-APNA; under conditions without catalase, 5-APNA was the main product in AncLLysO, whereas the compounds were hardly produced in AncLLysO2 (c in Figure ). On the contrary, the TPCA, which is generated by self-cyclizing the 2-keto-6-amino hexanoic acid (KH), was the main product in AncLysO2 judging from the concentration of other products, 5-APNA and 5-APNM (b and c in Figure ); the TPCA concentration could not be quantified because no standard compound is available. In a previous study, Trisrivirat et al reported that 5-APNA is generated by reacting the KH that is produced by hydrolyzing imino acid with in situ H 2 O 2 at the active site .…”

“…22 KH would be kept on the active site, and in situ H 2 O 2 can react with KH to produce 5-APNA. 22 Because of the difference in the substrate recognition mechanism, the KH may be remotely located from the FAD cofactor in AncLLysO, producing more 5-APNA than 5-APNM, as is the case for the reaction of L-Orn in L-LOX/ MOG. 21 Proposed Reaction Mechanism of AncLLysO2.…”