6isr
structure of lipase mutant with Cys-His-Asp catalytic triad
Structural highlights
FunctionLIPB_PSEA2 Hydrolysis of triglycerides. Is very stereospecific both in hydrolysis and in organic synthesis and has a potentially important application in glucolipid synthesis. Publication Abstract from PubMedEngineering 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. Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution.,Cen Y, Singh W, Arkin M, Moody TS, Huang M, Zhou J, Wu Q, Reetz MT Nat Commun. 2019 Jul 19;10(1):3198. doi: 10.1038/s41467-019-11155-3. PMID:31324776[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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