Structural and functional analysis of l-methionine oxidase identified through sequence data mining.

l-Amino acid oxidase (LAAO), an FAD-dependent enzyme, catalyzes the oxidation of l-amino acids (l-AAs) to their corresponding imino acids. While LAAOs, which can oxidize charged or aromatic l-AAs specifically, have been extensively characterized across various species, LAAOs that have high specificity toward alkyl-chain l-AAs, such as l-Met, are hardly characterized for now. In this study, we screened a highly specific l-Met oxidizing LAAOs from Burkholderiales bacterium (BbMetOx) and Undibacterium sp. KW1 (UndMetOx) using sequence similarity network (SSN) analysis. These enzymes displayed an order of magnitude higher specific activity towards l-Met compared to other l-AAs. Enzyme activity assays showed that these LAAOs operate optimally at moderate condition because the optimal pH and Tm values were pH 7.0 and 58-60°C. We determined the crystal structures of wild-type BbMetOx (BbMetOx(WT)) and an inactivated mutant, BbMetOx (K304A), at 2.7 Å and 2.2 Å resolution, respectively. The overall structure of BbMetOx is closely similar to other known LAAOs of which structures were determined. Comparative analysis of the BbMetOx structures revealed significant conformational changes in the catalytic domain, particularly a movement of approximately 8 Å in the Cα atom of residue Y180. Further analysis highlighted four residues, i.e., Y180, M182, F300, and M302, as critical for l-Met recognition, with alanine substitution at these positions resulting in loss of activity. This study not only underscores the utility of SSN for discovering novel LAAOs but also advances our understanding of substrate specificity in this enzyme family.

Turbo spin-echo-based enhanced acceleration-selective arterial spin labeling without electrocardiography or peripheral pulse unit triggering and contrast enhancement for lower extremity MRA.

PURPOSE: Lower extremity magnetic resonance angiography (MRA) without electrocardiography (ECG) or peripheral pulse unit (PPU) triggering and contrast enhancement is beneficial for diagnosing peripheral arterial disease (PAD) while avoiding synchronization failure and nephrogenic systemic fibrosis. This study aimed to compare the diagnostic performance of turbo spin-echo-based enhanced acceleration-selective arterial spin labeling (eAccASL) (TSE-Acc) of the lower extremities with that of turbo field-echo-based eAccASL (TFE-Acc) and triggered angiography non-contrast enhanced (TRANCE). METHODS: Nine healthy volunteers and a patient with PAD were examined on a 3.0 Tesla magnetic resonance imaging (MRI) system. The artery-to-muscle signal intensity ratio (SIR) and contrast-to-noise ratio (CNR) were calculated. The arterial visibility (1: poor, 4: excellent) and artifact contamination (1: severe, 4: no) were independently assessed by two radiologists. Phase-contrast MRI and digital subtraction angiography were referenced in a patient with PAD. Friedman's test and a post-hoc test according to the Bonferroni-adjusted Wilcoxon signed-rank test were used for the SIR, CNR, and visual assessment. p < 0.05 was considered statistically significant. RESULTS: No significant differences in nearly all the SIRs were observed among the three MRA methods. Higher CNRs were observed with TSE-Acc than those with TFE-Acc (anterior tibial artery, p = 0.014; peroneal artery, p = 0.029; and posterior tibial artery, p = 0.014) in distal arterial segments; however, no significant differences were observed upon comparison with TRANCE (all p > 0.05). The arterial visibility scores exhibited similar trends as the CNRs. The artifact contamination scores with TSE-Acc were significantly lower (but within an acceptable level) compared to those with TFE-Acc. In the patient with PAD, the sluggish peripheral arteries were better visualized using TSE-Acc than those using TFE-Acc, and the collateral and stenosis arteries were better visualized using TSE-Acc than those using TRANCE. CONCLUSION: Peripheral arterial visualization was better with TSE-Acc than that with TFE-Acc in lower extremity MRA without ECG or PPU triggering and contrast enhancement, which was comparable with TRANCE as the reference standard. Furthermore, TSE-Acc may propose satisfactory diagnostic performance for diagnosing PAD in patients with arrhythmia and chronic kidney disease.

Structural and functional analysis of hyper-thermostable ancestral L-amino acid oxidase that can convert Trp derivatives to D-forms by chemoenzymatic reaction.

Production of D-amino acids (D-AAs) on a large-scale enables to provide precursors of peptide therapeutics. In this study, we designed a novel L-amino acid oxidase, HTAncLAAO2, by ancestral sequence reconstruction, exhibiting high thermostability and long-term stability. The crystal structure of HTAncLAAO2 was determined at 2.2 Å by X-ray crystallography, revealing that the enzyme has an octameric form like a "ninja-star" feature. Enzymatic property analysis demonstrated that HTAncLAAO2 exhibits three-order larger kcat/Km values towards four L-AAs (L-Phe, L-Leu, L-Met, and L-Ile) than that of L-Trp. Through screening the variants, we obtained the HTAncLAAO2(W220A) variant, which shows a > 6-fold increase in kcat value toward L-Trp compared to the original enzyme. This variant applies to synthesizing enantio-pure D-Trp derivatives from L- or rac-forms at a preparative scale. Given its excellent properties, HTAncLAAO2 would be a starting point for designing novel oxidases with high activity toward various amines and AAs.

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

The flavin-dependent amine oxidase superfamily contains various l-amino acid oxidases (LAAOs) bearing different substrate specificities and enzymatic properties. LAAOs catalyze the oxidation of the α-amino group of l-amino acids (L-AAs) to produce imino acids and H2O2. In this study, an ancestral l-Lys α-oxidase (AncLLysO2) was designed utilizing genome-mined sequences from the Caulobacter species. The AncLLysO2 exhibited high specificity toward l-Lys; the k cat/K m values toward l-Lys were one and two orders larger than those of l-Arg and l-ornithine, respectively. Liquid chromatography-high resolution mass spectrometry analysis indicated that AncLLysO2 released imino acid immediately from the active site after completion of oxidation of the α-amino group. Crystal structures of the ligand-free, l-Lys- and l-Arg-bound forms of AncLLysO2 were determined at 1.4-1.6 Å resolution, indicating that the active site of AncLLysO2 kept an open state during the reaction and more likely to release products. The structures also indicated the substrate recognition mechanism of AncLLysO2; ε-amino, α-amino, and carboxyl groups of l-Lys formed interactions with Q357, A551, and R77, respectively. Biochemical and molecular dynamics simulation analysis of AncLLysO2 indicated that active site residues that indirectly interact with the substrate are also important to exhibit high activity; for example, the aromatic group of Y219 is important to ensure that the l-Lys substrate is placed in the correct position to allow the reaction to proceed efficiently. Taken together, we propose the reaction mechanism of AncLLysO2.