Pierce Colin T, Greenberg Lauren R, Walsh Meghan E, Shi Ke, Magee Drenen J, Aihara Hideki, Gordon Wendy, Evans Robert L, Kazlauskas Romas J
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA.
Crown College, Saint Bonifacius, Minnesota, USA.
Acta Crystallogr F Struct Biol Commun. 2025 Sep 1;81(Pt 9):398-405. doi: 10.1107/S2053230X25007034. Epub 2025 Aug 27.
The α/β-hydrolase fold superfamily includes esterases and hydroxynitrile lyases which, despite catalyzing different reactions, share a Ser-His-Asp catalytic triad. We report a 1.99 Å resolution crystal structure of HNL6V, an engineered variant of hydroxynitrile lyase from Hevea brasiliensis (HbHNL) containing seven amino-acid substitutions (T11G, E79H, C81L, H103V, N104A, G176S and K236M). The structure reveals that HNL6V maintains the characteristic α/β-hydrolase fold while exhibiting systematic shifts in backbone and catalytic atom positions. Compared with wild-type HbHNL, the C positions in HNL6V differ by a mean of 0.2 ± 0.1 Å, representing a statistically significant displacement. Importantly, the catalytic triad and oxyanion-hole atoms have moved 0.2-0.8 Å closer to their corresponding positions in SABP2, although they remain 0.3-1.1 Å from fully achieving the configuration of SABP2. The substitutions also increase local flexibility, particularly in the lid domain covering the active site. This structural characterization demonstrates that targeted amino-acid substitutions can systematically shift catalytic geometries towards those of evolutionarily related enzymes.
α/β-水解酶折叠超家族包括酯酶和羟基腈裂解酶，尽管它们催化不同的反应，但共享一个丝氨酸-组氨酸-天冬氨酸催化三联体。我们报道了HNL6V的晶体结构，分辨率为1.99 Å，它是巴西橡胶树羟基腈裂解酶（HbHNL）的工程变体，含有七个氨基酸取代（T11G、E79H、C81L、H103V、N104A、G176S和K236M）。该结构表明，HNL6V保持了典型的α/β-水解酶折叠，同时在主链和催化原子位置上呈现出系统性的位移。与野生型HbHNL相比，HNL6V中的C位置平均相差0.2±0.1 Å，这代表着具有统计学意义的位移。重要的是，催化三联体和氧阴离子空穴原子已向它们在SABP2中的相应位置移动了0.2 - 0.8 Å，尽管它们距离完全达到SABP2的构型仍有0.3 - 1.1 Å。这些取代还增加了局部灵活性，特别是在覆盖活性位点的盖子结构域。这种结构表征表明，靶向氨基酸取代可以使催化几何结构朝着进化相关酶的方向系统性地转变。
