Discovery of a regioselectivity switch in nitrating P450s guided by molecular dynamics simulations and Markov models.
The dynamic motions of protein structural elements, particularly flexible loops, are intimately linked with diverse aspects of enzyme catalysis. Engineering of these loop regions can alter protein stability, substrate binding and even dramatically impact enzyme function. When these flexible regions are unresolvable structurally, computational reconstruction in combination with large-scale molecular dynamics simulations can be used to guide the engineering strategy. Here we present a collaborative approach that consists of both experiment and computation and led to the discovery of a single mutation in the F/G loop of the nitrating cytochrome P450 TxtE that simultaneously controls loop dynamics and completely shifts the enzyme's regioselectivity from the C4 to the C5 position of L-tryptophan. Furthermore, we find that this loop mutation is naturally present in a subset of homologous nitrating P450s and confirm that these uncharacterized enzymes exclusively produce 5-nitro-L-tryptophan, a previously unknown biosynthetic intermediate.
Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, California 91125, USA.
Sheel C
SC
Dodani
Department of Chemistry, SIMBIOS NIH Center for Biomedical Computation, and Center for Molecular Analysis and Design, Stanford University, 318 Campus Drive, Stanford, California 94305, USA.
Gert
G
Kiss
Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, California 91125, USA.
Jackson K B
JK
Cahn
Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, California 91125, USA.
Ye
Y
Su
Department of Chemistry, SIMBIOS NIH Center for Biomedical Computation, and Center for Molecular Analysis and Design, Stanford University, 318 Campus Drive, Stanford, California 94305, USA.
Vijay S
VS
Pande
Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, California 91125, USA.
Frances H
FH
Arnold
D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics; D006418:Heme / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D008390:Markov Chains; D056004:Molecular Dynamics Simulation; D016296:Mutagenesis; D011487:Protein Conformation; D013237:Stereoisomerism; D013302:Streptomyces / Q000201:enzymology; D014364:Tryptophan / Q000737:chemistry
D006418:Heme; D006639:Histidine; D014364:Tryptophan; D003577:Cytochrome P-450 Enzyme System