6ISR

structure of lipase mutant with Cys-His-Asp catalytic triad

Summary for 6ISR

• Entry DOI: 10.2210/pdb6isr/pdb
• Descriptor: Lipase B, NICKEL (II) ION, DI(HYDROXYETHYL)ETHER, ... (5 entities in total)
• Functional Keywords: calb, hydrolase
• Biological source: Pseudozyma antarctica (Yeast)
• Total number of polymer chains: 2
• Total formula weight: 67707.67

Authors

Cen, Y.X.,Zhou, J.H.,Wu, Q. (deposition date: 2018-11-18, release date: 2019-07-24, Last modification date: 2024-10-23)

Primary citation

Cen, Y.,Singh, W.,Arkin, M.,Moody, T.S.,Huang, M.,Zhou, J.,Wu, Q.,Reetz, M.T.
Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution.
Nat Commun, 10:3198-3198, 2019

PubMed Abstract: Engineering artificial enzymes with high activity and catalytic mechanism different from naturally occurring enzymes is a challenge in protein design. For example, many attempts have been made to obtain active hydrolases by introducing a Ser → Cys exchange at the respective catalytic triads, but this generally induced a breakdown of activity. We now report that this long-standing dogma no longer pertains, provided additional mutations are introduced by directed evolution. By employing Candida antarctica lipase B (CALB) as the model enzyme with the Ser-His-Asp catalytic triad, a highly active cysteine-lipase having a Cys-His-Asp catalytic triad and additional mutations W104V/A281Y/A282Y/V149G can be evolved, showing a 40-fold higher catalytic efficiency than wild-type CALB in the hydrolysis of 4-nitrophenyl benzoate, and tolerating bulky substrates. Crystal structures, kinetics, MD simulations and QM/MM calculations reveal dynamic features and explain all results, including the preference of a two-step mechanism involving the zwitterionic pair Cys105/His224 rather than a concerted process.

PubMed: 31324776
DOI: 10.1038/s41467-019-11155-3

Experimental method

X-RAY DIFFRACTION (2.6 Å)