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1.
J Am Chem Soc ; 140(24): 7441-7444, 2018 06 20.
Article in English | MEDLINE | ID: mdl-29842777

ABSTRACT

Cells survive fluctuations in osmolality by accumulating and depleting highly soluble, usually neutral, small organic compounds. Natural selection has converged on a small set of such molecules, called osmolytes. The biophysical characterization of osmolytes, with respect to proteins, has centered on tertiary structure stability. Data about their effect on protein assemblies, whose formation is driven by surface interactions, is lacking. Here, we investigate the effects of osmolytes and related molecules on the stabilities of a protein and a protein complex. The results demonstrate that osmolytes are not differentiated from other cosolutes by their stabilizing influences on protein tertiary structure but by their compatibility with the interactions between protein surfaces that organize the cellular interior.


Subject(s)
Bacterial Proteins/metabolism , Drosophila Proteins/metabolism , Organic Chemicals/metabolism , Animals , Bacterial Proteins/chemistry , Drosophila/chemistry , Drosophila Proteins/chemistry , Organic Chemicals/chemistry , Osmolar Concentration , Protein Binding , Protein Multimerization , Static Electricity , Streptococcus/chemistry , src Homology Domains
2.
Biotechnol Bioeng ; 115(6): 1416-1426, 2018 06.
Article in English | MEDLINE | ID: mdl-29460311

ABSTRACT

Engineered cytochrome P450s are emerging as powerful synthetic tools due to their ability catalyze non-native metallocarbenoid and -nitrenoid insertion reactions. P450-mediated cyclopropanation has garnered particular interest due to the high selectivity demonstrated by engineered scaffolds and their application towards the synthesis of therapeutic agents. We previously reported that mutation of a conserved, first-shell heme-ligating Cys to Ser led to significant improvements in cyclopropanation activity in a model enzyme, P450BM3h . Here, we demonstrate that mutation of a ubiquitously conserved second-shell Phe (F393) to His or Ala, provides complementary increases in the P450 heme reduction potential and conversion to cyclopropanation products when compared to first-shell Cys to Ser mutations. Furthermore, we show that these mutations confer improved non-natural catalysis in 4 diverse P450 scaffolds.


Subject(s)
Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation, Missense , Cyclopropanes/metabolism , Heme/metabolism , Oxidation-Reduction , Protein Engineering , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
4.
Chembiochem ; 17(5): 394-7, 2016 Mar 02.
Article in English | MEDLINE | ID: mdl-26690878

ABSTRACT

Cytochrome P450s and other heme-containing proteins have recently been shown to have promiscuous activity for the cyclopropanation of olefins using diazoacetate reagents. Despite the progress made thus far, engineering selective catalysts for all possible stereoisomers for the cyclopropanation reaction remains a considerable challenge. Previous investigations of a model P450 (P450BM3 ) revealed that mutation of a conserved active site threonine (Thr268) to alanine transformed the enzyme into a highly active and selective cyclopropanation catalyst. By incorporating this mutation into a diverse panel of P450 scaffolds, we were able to quickly identify enantioselective catalysts for all possible diastereomers in the model reaction of styrene with ethyl diazoacetate. Some alanine variants exhibited selectivities that were markedly different from the wild-type enzyme, with a few possessing moderate to high diastereoselectivity and enantioselectivities up to 97 % for synthetically challenging cis-cyclopropane diastereomers.


Subject(s)
Alkenes/chemistry , Conserved Sequence , Cyclopropanes/chemistry , Cytochrome P-450 Enzyme System/genetics , Mutation , Catalytic Domain , Cytochrome P-450 Enzyme System/chemistry , Stereoisomerism
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