Journal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Volume 499, Issue 2, Pages 233-238Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2018.03.134
Keywords
Loop engineering; Substrate affinity; Loop flexibility; Active-site-decorating loops; Arginine deiminase
Categories
Funding
- National Natural Science Foundation of China [31700693, 21708046]
- Zhejiang Provincial Natural Science Foundation [LQ17C050002]
- Chinese Academy of Sciences
- 'Thousand Talents Program' of Tianjin
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Protein engineering of enzyme loop regions is an effective strategy to improve enzymatic properties. Previous studies that aimed to boost the activity of PpADI (an arginine deiminase from Pseudomonas plecoglossicida) under physiological conditions yielded several significantly improved variants that harbor substitutions predominantly located in active-site-decorating loops. A multi-site saturation mutagenesis at four positions in loop 1 (37, 38, 42, and 43) and three positions in loop 4 (402, 403, and 404) was performed to elucidate the importance of these loops in modulating the substrate affinity of PpADI. The identified best variant (M6-L1-4) showed a decreased Sas ('K-M') of OAS mM compared with the parent M6 (0.81 mM). Subsequently, a rational design to recombine beneficial substitutions within loops 1 and 4 yielded variant L6 with a substantially decreased S-0.5 value (0.17 mM). A comprehensive simulation analysis resulted in a conclusion that high loop flexibility (especially the gating residue Arg400) is beneficial for substrate affinity due to less efficient blocking of the active site. (C) 2018 Elsevier Inc. All rights reserved.
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