3NYD

Crystal Structure of Kemp Eliminase HG-2 Complexed with Transition State Analog 5-Nitro Benzotriazole

3NYD の概要

• エントリーDOI: 10.2210/pdb3nyd/pdb
• 分子名称: Endo-1,4-beta-xylanase, 5-nitro-1H-benzotriazole, ACETATE ION, ... (5 entities in total)
• 機能のキーワード: tim barrel, kemp elimination enzyme, hydrolase
• 由来する生物種: Thermoascus aurantiacus
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 69295.43

構造登録者

Lee, T.M.,Privett, H.K.,Kaiser, J.T.,Mayo, S.L. (登録日: 2010-07-14, 公開日: 2011-06-29, 最終更新日: 2024-10-30)

主引用文献

Privett, H.K.,Kiss, G.,Lee, T.M.,Blomberg, R.,Chica, R.A.,Thomas, L.M.,Hilvert, D.,Houk, K.N.,Mayo, S.L.
Iterative approach to computational enzyme design.
Proc.Natl.Acad.Sci.USA, 109:3790-3795, 2012

PubMed Abstract: A general approach for the computational design of enzymes to catalyze arbitrary reactions is a goal at the forefront of the field of protein design. Recently, computationally designed enzymes have been produced for three chemical reactions through the synthesis and screening of a large number of variants. Here, we present an iterative approach that has led to the development of the most catalytically efficient computationally designed enzyme for the Kemp elimination to date. Previously established computational techniques were used to generate an initial design, HG-1, which was catalytically inactive. Analysis of HG-1 with molecular dynamics simulations (MD) and X-ray crystallography indicated that the inactivity might be due to bound waters and high flexibility of residues within the active site. This analysis guided changes to our design procedure, moved the design deeper into the interior of the protein, and resulted in an active Kemp eliminase, HG-2. The cocrystal structure of this enzyme with a transition state analog (TSA) revealed that the TSA was bound in the active site, interacted with the intended catalytic base in a catalytically relevant manner, but was flipped relative to the design model. MD analysis of HG-2 led to an additional point mutation, HG-3, that produced a further threefold improvement in activity. This iterative approach to computational enzyme design, including detailed MD and structural analysis of both active and inactive designs, promises a more complete understanding of the underlying principles of enzymatic catalysis and furthers progress toward reliably producing active enzymes.

PubMed: 22357762
DOI: 10.1073/pnas.1118082108

実験手法

X-RAY DIFFRACTION (1.23 Å)