Abstract
The acyl binding site of Rhizopus delemar prolipase and mature lipase was altered through site-directed mutagenesis to improve lipase specificity for short-or medium-chain length fatty acids. Computer-generated structural models of R. delemar lipase were used in mutant protein design and in the interpretation of the catalytic properties of the resulting recombinant enzymes. Molecular dynamics simulations of the double mutant, val209trp+phe112trp, predicted that the introduction of trp112 and trp209 in the acyl binding groove would sterically hinder the docking of fatty acids longer than butyric acid. Assayed against a mixture of triacylglycerol substrates, the val209trp+phe112trp mature lipase mutant showed an 80-fold increase in the hydrolysis of tributyrin relative to the hydrolysis of tricaprylin while no triolein hydrolysis was detected. By comparison, the val94Trp mutant, predicted to pose steric or geometric constraints for docking fatty acids longer than caprylic acid in the acyl binding groove, resulted in a modest 1.4-fold increase in tricaprylin hydrolysis relative to the hydrolysis of tributyrin. Molecular models of the double mutant phe95asp+phe214arg indicated the creation of a salt bridge between asp95 and arg214 across the distal end of the acyl binding groove. When challenged with a mixture of triacylglycerols, the phe95asp+phe214arg substitutions resulted in an enzyme with 3-fold enhanced relative activity for tricaprylin compared to triolein, suggesting that structural determinants for medium-chain length specificity may reside in the distal end of the acyl binding groove. Attempts to introduce a salt bridge within 8 Å of the active site by the double mutation leu146lys+ser115asp destroyed catalytic activity entirely. Similarly, the substitution of polar Gln at the rim of the acyl binding groove for phe 112 largely eliminated catalytic activity of the lipase.
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Abbreviations
- cDNA:
-
complementary DNA
- IPTG:
-
isopropyl-b-d-thiogalactoside
- Phe:
-
phenylalanine
- Rd :
-
Rhizopus delemar
- TB:
-
tributyrin
- TC:
-
tricaprylin
- TL:
-
trilaurin
- TO:
-
triolein
- Tris:
-
ris(hydroxymethyl)aminomethane
- val:
-
valine
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Klein, R.R., King, G., Moreau, R.A. et al. Altered acyl chain length specificity of Rhizopus delemar lipase through mutagenesis and molecular modeling. Lipids 32, 123–130 (1997). https://doi.org/10.1007/s11745-997-0016-1
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DOI: https://doi.org/10.1007/s11745-997-0016-1