Global incorporation of norleucine in place of methionine in cytochrome P450 BM‐3 heme domain increases peroxygenase activity

  title={Global incorporation of norleucine in place of methionine in cytochrome P450 BM‐3 heme domain increases peroxygenase activity},
  author={Patrick C Cirino and Yi Tang and Katsuyuki Takahashi and David A. Tirrell and Frances H. Arnold},
  journal={Biotechnology and Bioengineering},
In this study we have replaced all 13 methionine residues in the cytochrome P450 BM‐3 heme domain (463 amino acids) with the isosteric methionine analog norleucine. This experiment has provided a means of testing the functional limits of globally incorporating into an enzyme an unnatural amino acid in place of its natural analog, and also an efficient way to test whether inactivation during peroxide‐driven P450 catalysis involves methionine oxidation. Although there was no increase in the… 
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Circular dichroism studies reveal that although fluorination greatly improves the secondary structure of CAT, a large structural destabilization upon increased levels of TFL incorporation occurs at elevated temperatures, suggesting that enhanced secondary structure afforded by TFL incorporate does not necessarily lead to an improvement in stability.
Enhancing the Efficiency and Regioselectivity of P450 Oxidation Catalysts by Unnatural Amino Acid Mutagenesis
The development of effective strategies for modulating the reactivity and selectivity of cytochrome P450 enzymes represents a key step toward expediting the use of these biocatalysts for synthetic
Residue-Specific Incorporation of the Non-Canonical Amino Acid Norleucine Improves Lipase Activity on Synthetic Polyesters
Interestingly, a 50% increased activity was found for TTL [Nle] towards ionic phthalic polyesters containing different ether diols compared to the parent enzyme TTL [Met], demonstrating the high potential of non-canonical amino acids for enzyme engineering.
A catalytic role for methionine revealed by a combination of computation and experiments on phosphite dehydrogenase
Combined quantum mechanics/molecular mechanics simulations of the reaction catalysed by phosphite dehydrogenase (PTDH) andalyses of the Protein Data Bank and Cambridge Structural Database indicate that this type of interaction may be relatively widespread, with implications for enzyme-catalysed reaction mechanisms and protein structure.
P450(BM3) (CYP102A1): connecting the dots.
This review draws together the disparate research themes of P450(BM3) (CYP102A1) and places them in a historical context with the aim of creating a resource that can be used as a gateway to the field.
On the roles of methionine and the importance of its microenvironments in redox metalloproteins.
While the importance of interactions between Met and other groups is established in biological systems, less is known about their roles in redox metalloproteins and the view is that this is an area that is ready for greater attention.
Manipulation of enzyme properties by noncanonical amino acid incorporation
Considering the generality of NCAAI incorporation, it is expected its application could be expanded to improve other enzyme properties, such as substrate specificity and solvent resistance in the near future.


Regioselectivity and Activity of Cytochrome P450 BM‐3 and Mutant F87A in Reactions Driven by Hydrogen Peroxide
While significantly enhancing peroxygenase activity, the F87A mutation also shifts hydroxylation further away from the terminal position, and the H2O2- driven reactions with either the full-length BM-3 enzyme or the heme domain are slow, but yield product distributions very similar to those generated when using NADPH and O2.
Efficient incorporation of unsaturated methionine analogues into proteins in vivo
Results of the in vitro assays corroborate the in vivo incorporation results, suggesting that success or failure of analogue incorporation in vivo is controlled by MetRS.
The use of t-butyl hydroperoxide as a probe for methionine oxidation in proteins.
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  • 1996
The susceptibility of native recombinant interferon gamma and recombinant tissue-type plasminogen activator to methionine oxidation when treated with the oxidizing agent t-butyl hydroperoxide (TBHP) was investigated and showed that during treatment of a native protein with TBHP only the exposed methamphetamineionine residues were oxidized.
Substitution of the methionine residues of calmodulin with the unnatural amino acid analogs ethionine and norleucine: Biochemical and spectroscopic studies
  • T. Yuan, H. Vogel
  • Chemistry, Biology
    Protein science : a publication of the Protein Society
  • 1999
Biosynthetically substituted the unnatural amino acids ethionine (Eth) and norleucine (Nle) for the nine Met residues of CaM, showing that the “active site” Met residuesof CaM play a distinct role in the activation of different target enzymes, in agreement with site‐directed mutagenesis studies of the Met residuesOf CaM.
Comparing the effect on protein stability of methionine oxidation versus mutagenesis: steps toward engineering oxidative resistance in proteins.
Compared with the effects of substituting methionines with isoleucine and leucine, a potential strategy for stabilizing proteins against oxidative damage, a simple substitution mutagenesis strategy to protect a protein against oxidative destabilization is practical for some methionine residues.
A continuous spectrophotometric assay for P450 BM-3, a fatty acid hydroxylating enzyme, and its mutant F87A.
A rapid and continuous spectrophotometric activity assay for cytochrome P450 BM-3 based on the conversion of p-nitrophenoxycarboxylic acids (pNCA) to omega-oxycaroxydodecanoic and the chromophore p-Nitrophenolate was developed.
The effect on subtilisin activity of oxidizing a methionine residue.
Engineering an enzyme by site-directed mutagenesis to be resistant to chemical oxidation.
Probing the heme iron coordination structure of pressure-induced cytochrome P420cam.
Spectral evidence and results from a parallel investigation of the spectroscopically related inactive form of chloroperoxidase lead to the conclusion that a sulfur-derived proximal ligand is coordinated to the heme of ferric cytochrome P420cam.
Oxidation of methionyl residues in proteins: tools, targets, and reversal.
  • W. Vogt
  • Chemistry, Biology
    Free radical biology & medicine
  • 1995