Structural Characterization of Sphingomonas sp. KT-1 PahZ1-Catalyzed Biodegradation of Thermally Synthesized Poly(aspartic acid)
Structural Characterization of Sphingomonas sp. KT-1 PahZ1-Catalyzed Biodegradation of Thermally Synthesized Poly(aspartic acid)
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DOI：
10.1021/acssuschemeng.0c01158
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发表时间：
2020-07-27
影响因子：
8.4
通讯作者：
Weiland, Mitch H.
中科院分区：
文献类型：
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作者：
Brambley, Chad A.;Bolay, Austin L.;Weiland, Mitch H.
聚天冬氨酸（PAA）是不可生物降解聚羧酸酯的绿色替代品，在工业和生物医学环境中都有应用。PAA是通过加热单体天冬氨酸制得聚琥珀胺，聚琥珀胺可以开环制得由30% -酰胺和70% -酰胺键组成的热PAA。本文报道了细菌鞘氨单胞菌KT-1 （PahZ1(KT-1)）的PAA水解酶的第一个x射线晶体结构，该酶通过选择性切割ss-酰胺键将合成的PAA降解为低聚（天冬氨酸）。该结构被解析为2.45埃，显示出一个二聚体组装，其中每个单体都保持一个α /ss水解酶折叠，并有一个突出的、正的内衬槽，负责结合阴离子聚合物底物。每个单体的假定催化位点位于酶的相对表面上。小角度x射线散射/多角度光散射数据表明，二聚体界面主要是疏水性的，并通过侧面氢键进一步稳定。分子动力学模拟支持先前确定的通过使底物与活性位点Ser对齐的构象变化，仅ss-酰胺键的特异性裂解。这些数据为进一步了解PAA水解的机制提供了一个框架，并为利用蛋白质工程催化其他异种生物降解开辟了机会。
Poly(aspartic acid) (PAA) is a green alternative to non-biodegradable poly(carboxylates) and has applications in both industrial and biomedical settings. PAA is synthesized by heating monomeric aspartic acid to yield a polysuccinamide that can be ring-opened to yield thermal PAA composed of 30% alpha-amide and 70% ss-amide linkages. Here, we report the first X-ray crystal structure of a PAA hydrolase from the bacteria Sphingomonas sp. KT-1 (PahZ1(KT-1)) which functions to degrade synthetic PAA to oligo(aspartic acid) by selective cleavage of ss-amide linkages. The structure was solved to 2.45 angstrom and shows a dimeric assembly where each monomer maintains an alpha/ss hydrolase fold with a prominent, positively lined trough responsible for binding the anionic polymeric substrate. The putative catalytic sites of each monomer lie at the surface of the enzyme on opposite faces. The dimeric interface, as supported by small-angle X-ray scattering/multi-angle light scattering data, is primarily hydrophobic and is further stabilized by flanking hydrogen bonds. Molecular dynamics simulations support the previously determined specific cleavage of only the ss-amide linkage through a conformational change that aligns the substrate with the active site Ser. These data provide a scaffold for further understanding the mechanism of PAA hydrolysis and opens the opportunity for using protein engineering to catalyze the biodegradation of other xenobiotics.