We have determined the x-ray crystal structure of l-lysine ε-oxidase from Marinomonas mediterranea in its native and l-lysine-complex forms at 1.94- and 1.99-Å resolution, respectively. In the native enzyme, electron densities clearly indicate the presence of cysteine tryptophylquinone (CTQ) previously identified in quinohemoprotein amine dehydrogenase. In the l-lysine-complex, an electron density corresponding to the bound l-lysine shows that its ε-amino group is attached to the C6 carbonyl group of CTQ, suggesting the formation of a Schiff-base intermediate. Collectively, the present crystal structure provides the first example of an enzyme employing a tryptophylquinone cofactor in an amine oxidase.

Insolubility of proteins expressed in the Escherichia coli expression system hinders the progress of both basic and applied research. Insoluble proteins contain residues that decrease their solubility (aggregation hotspots). Mutating these hotspots to optimal amino acids is expected to improve protein solubility. To date, however, the identification of these hotspots has proven difficult. In this study, using a combination of approaches involving directed evolution and primary sequence analysis, we found two rules to help inductively identify hotspots: the α-helix rule, which focuses on the hydrophobicity of amino acids in the α-helix structure, and the hydropathy contradiction rule, which focuses on the difference in hydrophobicity relative to the corresponding amino acid in the consensus protein. By properly applying these two rules, we succeeded in improving the probability that expressed proteins would be soluble. Our methods should facilitate research on various insoluble proteins that were previously difficult to study due to their low solubility.

In this study, it was shown for the first time that l‐amino acid oxidase of Pseudomonas sp. AIU813, renamed as l‐amino acid oxidase/monooxygenase (l‐AAO/MOG), exhibits l‐lysine 2‐monooxygenase as well as oxidase activity. l‐Lysine oxidase activity of l‐AAO/MOG was increased in a p‐chloromercuribenzoate (p‐CMB) concentration‐dependent manner to a final level that was five fold higher than that of the non‐treated enzyme. In order to explain the effects of modification by the sulfhydryl reagent, saturation mutagenesis studies were carried out on five cysteine residues, and we succeeded in identifying l‐AAO/MOG C254I mutant enzyme, which showed five‐times higher specific activity of oxidase activity than that of wild type. The monooxygenase activity shown by the C254I variant was decreased significantly. Moreover, we also determined a high‐resolution three‐dimensional structure of l‐AAO/MOG to provide a structural basis for its biochemical characteristics. The key residue for the activity conversion of l‐AAO/MOG, Cys‐254, is located near the aromatic cage (Trp‐418, Phe‐473, and Trp‐516). Although the location of Cys‐254 indicates that it is not directly involved in the substrate binding, the chemical modification by p‐CMB or C254I mutation would have a significant impact on the substrate binding via the side chain of Trp‐516. It is suggested that a slight difference of the binding position of a substrate can dictate the activity of this type of enzyme as oxidase or monooxygenase.

A large number of protein sequences are registered in public databases such as PubMed. Functionally uncharacterized enzymes are included in these databases, some of which likely have potential for industrial applications. However, assignment of the enzymes remained difficult tasks for now. In this study, we assigned a total of 28 original sequences to uncharacterized enzymes in the FAD-dependent oxidase family expressed in some species of bacteria including Chryseobacterium, Flavobacterium, and Pedobactor. Progenitor sequence of the assigned 28 sequences was generated by ancestral sequence reconstruction, and the generated sequence exhibited L-lysine oxidase activity; thus, we named the enzyme AncLLysO. Crystal structures of ligand-free and ligand-bound forms of AncLLysO were determined, indicating that the enzyme recognizes L-Lys by hydrogen bond formation with R76 and E383. The binding of L-Lys to AncLLysO induced dynamic structural change at a plug loop formed by residues 251 to 254. Biochemical assays of AncLLysO variants revealed the functional importance of these substrate recognition residues and the plug loop. R76A and E383D variants were also observed to lose their activity, and the k_cat/K_m value of G251P and Y253A mutations were approximately 800- to 1800-fold lower than that of AncLLysO, despite the indirect interaction of the substrates with the mutated residues. Taken together, our data demonstrate that combinational approaches to sequence classification from database and ancestral sequence reconstruction may be effective not only to find new enzymes using databases of unknown sequences but also to elucidate their functions.