Department of Chemistry, University of British Columbia, Vancouver, British Columbia, V6T 1Z1, Canada.
Angew Chem Int Ed Engl. 2018 Aug 27;57(35):11359-11364. doi: 10.1002/anie.201806792. Epub 2018 Aug 1.
Functional metagenomics has opened new opportunities for enzyme discovery. To exploit the full potential of this new tool, the design of selective screens is essential, especially when searching for rare enzymes. To identify novel glycosidases that employ cleavage strategies other than the conventional Koshland mechanisms, a suitable screen was needed. Focusing on the unsaturated glucuronidases (UGLs), it was found that use of simple aryl glycoside substrates did not allow sufficient discrimination against β-glucuronidases, which are widespread in bacteria. While conventional glycosidases cannot generally hydrolyze thioglycosides efficiently, UGLs follow a distinct mechanism that allows them to do so. Thus, fluorogenic thioglycoside substrates featuring thiol-based self-immolative linkers were synthesized and assessed as selective substrates. The generality of the approach was validated with another family of unconventional glycosidases, the GH4 enzymes. Finally, the utility of these substrates was tested by screening a small metagenomic library.
功能宏基因组学为酶的发现开辟了新的机会。为了充分利用这一新型工具，有必要设计有针对性的筛选方法，特别是在寻找稀有酶时。为了鉴定采用非常规科歇尔机制以外的切割策略的新型糖苷酶，需要设计合适的筛选方法。本研究聚焦于不饱和葡萄糖醛酸酶（UGL），发现使用简单的芳基糖苷底物并不能有效区分β-葡萄糖醛酸酶，因为β-葡萄糖醛酸酶在细菌中广泛存在。虽然常规糖苷酶通常不能有效地水解硫代糖苷，但 UGL 遵循一种独特的机制，使它们能够做到这一点。因此，合成了带有基于硫醇的自毁连接基团的荧光硫代糖苷底物，并将其评估为选择性底物。该方法的通用性已通过另一家族的非常规糖苷酶 GH4 酶得到验证。最后，通过筛选一个小型宏基因组文库来测试这些底物的实用性。
