Molecular and Spectroscopic Characterization of Aspergillus flavipes and Pseudomonas putida L-Methionine γ-Lyase in Vitro
Molecular and Spectroscopic Characterization of Aspergillus flavipes and Pseudomonas putida L-Methionine γ-Lyase in Vitro
复制标题
DOI：
10.1007/s12010-016-2299-x
复制
发表时间：
2017-04-01
影响因子：
3
通讯作者：
Esener, Sadik
中科院分区：
文献类型：
--
作者：
El-Sayed, Ashraf S. A.;Ruff, Laura E.;Esener, Sadik
恶臭假单胞菌L-甲硫氨酸γ-裂解酶（PpMGL）是一种高效的抗癌药物，但其抗原性和稳定性仍是其临床应用的关键问题。从我们的研究，曲霉黄柄L-蛋氨酸γ-裂解酶（AfMGL）显示出更实惠的生化特性比PpMGL。因此，本工作的目的是通过其内部醛亚胺键的稳定性、吡哆醛5 '-磷酸（PLP）的互变异构性和响应于物理化学因素的结构稳定性来比较评估AfMGL和PpMGL的功能特性。AfMGL和PpMGL的内部席夫碱对羟胺和人血清白蛋白具有相同的稳定性。酸性pH值导致强烈的分裂的内部席夫碱，诱导MGL的展开，相比，中性-碱性pH值。在λ 280 nm激发下，AfMGL和PpMGL在λ 335 nm处对固有色氨酸和λ 560 nm处对内部席夫碱具有相同的荧光发射光谱。在酸性pH下检测到酮烯胺到烯醇亚胺的最大PLP互变异构位移，导致完全的酶解折叠，亚基解离和内在PLP的互变异构位移，而不是中性-碱性的。通过DSF测定在热稳定剂/去稳定剂存在下AfMGL和PpMGL的T（m）。AfMGL和PpMGL的T（m）分别为73.1 A ° C和74.4 A ° C，表明与这两种酶的三级结构更接近。与盐酸胍和尿素相比，海藻糖和EDTA使AfMGL和PpMGL的T（m）略有增加。活性位点和PLP结合结构域在AfMGL和PpMGL中是相同保守的。
Pseudomonas putida L-methionine gamma-lyase (PpMGL) has been recognized as an efficient anticancer agent, however, its antigenicity and stability remain as critical challenges for its clinical use. From our studies, Aspergillus flavipes L-methionine gamma-lyase (AfMGL) displayed more affordable biochemical properties than PpMGL. Thus, the objective of this work was to comparatively assess the functional properties of AfMGL and PpMGL via stability of their internal aldimine linkage, tautomerism of pyridoxal 5'-phosphate (PLP) and structural stability responsive to physicochemical factors. The internal Schiff base of AfMGL and PpMGL have the same stability to hydroxylamine and human serum albumin. Acidic pHs resulted in strong cleavage of the internal Schiff base, inducing the unfolding of MGLs, compared to neutral-alkaline pHs. At lambda 280 nm excitation, both AfMGL and PpMGL have identical fluorescence emission spectra at lambda 335 nm for the intrinsic tryptophan and lambda 560 nm for the internal Schiff base. The maximum PLP tautomeric shift of ketoenamine to enolimine was detected at acidic pH causing complete enzyme unfolding, subunits dissociation and tautomeric shift of intrinsic PLP, rather than neutral-alkaline ones. The T (m) of AfMGL and PpMGL in presence of thermal stabilizer/ destabilizer was assayed by DSF. The T (m) of AfMGL and PpMGL was 73.1 A degrees C and 74.4 A degrees C, respectively, suggesting the higher proximity to the tertiary structure of both enzymes. The T (m) of AfMGL and PpMGL was slightly increased by trehalose and EDTA in contrast to guanidine HCl and urea. The active site and PLP-binding domains are identically conserved in both AfMGL and PpMGL.