Department of Biochemical Engineering, Beijing University of Chemical Technology, Beijing, China.
Department of Biochemical Engineering, Beijing University of Chemical Technology, Beijing, China.
Enzyme Microb Technol. 2019 Feb;121:8-16. doi: 10.1016/j.enzmictec.2018.10.013. Epub 2018 Oct 29.
3,4 Dihydroxy phenyl l-alanine (L-DOPA) is the gold standard Parkinson's disease therapy. A heme-dependent peroxidase (HDP) catalyzes the ortho-hydroxylation of l-tyrosine to l-DOPA using HO as the co-substrate. d-amino acid oxidase (DAAO) catalyzes the oxidative deamination of d-amino acids (e.g. d-alanine), and HO is evolved. However, both the enzymes DAAO and HDP can be inactivated by HO during the catalysis. In situ generation and utilization of HO can siginificatly reduce the inactivation by HO. HDP exists as a monomer and DAAO is a dimeric enzyme. Herein, the C-terminus of HDP was specifically ligated to the N-terminus of the DAAO subunit with native peptide through the in vivo monomer-subunit splicing. In the splicing product HDP&DAAO, HDP is close to the DAAO subunit at a molecular distance, and the transfer of HO from DAAO to HDP is facilitated. In addition, HDP&DAAO exhibited a higher stability than HDP. Kinetics analysis showed that both the substrates l-tyrosine and d-alanine obey the Michaelis-Menten kinetics. For the deamination of d-alanine, the catalytic efficiency of HDP&DAAO is 3.05 times that of DAAO. For the sybthesis of l-DOPA from l-tyrosine, the catalytic efficiency of HDP&DAAO is 1.58 times that of HDP. Furthermore, HDP&DAAO was encapsulated within a Znic-based coordination polymer (Zn-CP). The morphorogy of HDP&DAAO/Zn-CP can be regulated by the enzyme concentration, the catalytic efficiency of the conjugates was found to be dependent on the morphorogy. The conjugates HDP&DAAO/Zn-CP exhibited a higher catalytic efficiency than free HDP&DAAO.
3,4 二羟苯丙氨酸（L-DOPA）是治疗帕金森病的金标准。血红素依赖过氧化物酶（HDP）以 HO 为共底物，催化 l-酪氨酸的邻位羟化生成 L-DOPA。d-氨基酸氧化酶（DAAO）催化 d-氨基酸（如 d-丙氨酸）的氧化脱氨，产生 HO。然而，在催化过程中，HO 可以使 DAAO 和 HDP 失活。HO 的原位生成和利用可以显著减少 HO 的失活。HDP 以单体形式存在，DAAO 是二聚体酶。在此，通过体内单体-亚基拼接，将 HDP 的 C 末端通过天然肽特异性连接到 DAAO 亚基的 N 末端。在拼接产物 HDP&DAAO 中，HDP 与 DAAO 亚基在分子距离上接近，促进了 HO 从 DAAO 向 HDP 的转移。此外，HDP&DAAO 比 HDP 具有更高的稳定性。动力学分析表明，l-酪氨酸和 d-丙氨酸这两种底物均遵循米氏动力学。对于 d-丙氨酸的脱氨反应，HDP&DAAO 的催化效率是 DAAO 的 3.05 倍。对于 l-酪氨酸合成 L-DOPA，HDP&DAAO 的催化效率是 HDP 的 1.58 倍。此外，将 HDP&DAAO 包封在基于锌的配位聚合物（Zn-CP）中。通过酶浓度可以调节 HDP&DAAO/Zn-CP 的形态，发现缀合物的催化效率取决于形态。HDP&DAAO/Zn-CP 缀合物的催化效率高于游离 HDP&DAAO。
