l‐Lysine oxidase (LysOX) is a FAD‐dependent homodimeric enzyme that catalyzes the oxidative deamination of l‐lysine to produce α‐keto‐ε‐aminocaproate with ammonia and hydrogen peroxide. LysOX shows strict substrate specificity for l‐lysine, whereas most l‐amino acid oxidases (LAAOs) exhibit broad substrate specificity for l‐amino acids. Previous studies of LysOX showed that overall structural similarity to the well‐studied snake venom LAAOs. However, the molecular mechanism of strict specificity for l‐lysine was still unclear. We here determined the structure of LysOX in complex with l‐lysine at 1.7 Å resolution. The structure revealed that the hydrogen bonding network formed by D212, D315, and A440 with two water molecules is responsible for the recognition of the side chain amino group. In addition, a narrow hole formed by five hydrophobic residues in the active site contributes to strict substrate specificity. Mutation studies demonstrated that D212 and D315 are essential for l‐lysine recognition, and the D212A/D315A double mutant LysOX showed different substrate specificity from LysOX. Moreover, the structural basis of the substrate specificity change has also been revealed by the structural analysis of the mutant variant and its substrate complexes. These results clearly explain the molecular mechanism of the strict specificity of LysOX and suggest that LysOX is a potential candidate for a template to design LAAOs specific to other l‐amino acids.

中文翻译：

---

绿色木霉中 l-赖氨酸 α-氧化酶严格底物识别的结构基础。

l-赖氨酸氧化酶 (LysOX) 是一种依赖 FAD 的同型二聚酶，可催化l-赖氨酸的氧化脱氨，与氨和过氧化氢产生 α-酮-ε-氨基己酸酯。LysOX 对l-赖氨酸显示出严格的底物特异性，而大多数l-氨基酸氧化酶 (LAAO) 对l-氨基酸显示出广泛的底物特异性。先前对 LysOX 的研究表明，总体结构与经过充分研究的蛇毒 LAAO 相似。然而，严格的特异性的分子机制升赖氨酸仍不清楚。我们在这里确定了与l复合的 LysOX 的结构-赖氨酸，分辨率为 1.7 Å。该结构揭示了由D212、D315和A440与两个水分子形成的氢键网络负责侧链氨基的识别。此外，由活性位点中的五个疏水残基形成的窄孔有助于严格的底物特异性。突变研究表明 D212 和 D315 对l‐赖氨酸识别，D212A/D315A 双突变体 LysOX 显示出与 LysOX 不同的底物特异性。此外，突变变体及其底物复合物的结构分析也揭示了底物特异性变化的结构基础。这些结果清楚地解释了 LysOX 严格特异性的分子机制，并表明 LysOX 是设计其他l-氨基酸特异性 LAAO 的潜在模板候选者。