Enhanced Substrate Specificity of Thermostable Cytochrome P450 CYP175A1 by Site Saturation Mutation on Tyrosine 68

Karmakar, Srabani; Nag, Sudip Kumar; Taher, Mohd; Kansara, Bharat T.; Mazumdar, Shyamalava

doi:10.1007/s10930-022-10084-3

Cited by 2 publications

(1 citation statement)

References 28 publications

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“…7,8 The engineered cytochrome P450 enzymes were shown to be more catalytically active and were also capable of catalyzing 'new to nature' chemical reactions such as C-C coupling 9 and nitrogen insertion into an unactivated C-H bond. 10 The rational evolution of the enzymes, has mostly been focused on active site engineering using site-directed and saturation mutagenesis, 11 and also by using novel decoy molecules to expand the substrate scope. 12,13 Modication of the metal cofactor 14,15 along with active site mutation 16,17 has also been investigated to catalyze abiological reactions by using this enzyme.…”

Section: Introductionmentioning

confidence: 99%

Computationally guided bioengineering of the active site, substrate access pathway, and water channels of thermostable cytochrome P450, CYP175A1, for catalyzing the alkane hydroxylation reaction

Taher,

Dubey,

Mazumdar

2023

Chem. Sci.

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Section: Introductionmentioning

confidence: 99%

Computationally guided bioengineering of the active site, substrate access pathway, and water channels of thermostable cytochrome P450, CYP175A1, for catalyzing the alkane hydroxylation reaction

Taher,

Dubey,

Mazumdar

2023

Chem. Sci.

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Details matter: Defining substrate specificity in arogenate versus prephenate dehydratases

Clayton,

Smith-Uffen,

Duennwald

et al. 2024

Preprint

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Phenylalanine is an essential aromatic amino acid that can only be synthesized de novo by microorganisms and plants. In microorganisms, phenylalanine is synthesized through the prephenate pathway, requiring the activity of a prephenate dehydratase (PDT). In plants, phenylalanine is synthesized instead mostly through the arogenate pathway, requiring the enzyme arogenate dehydratase (ADT). In Arabidopsis, there is a family of six ADTs that catalyze this final step of phenylalanine biosynthesis. However, two of the AtADTs, AtADT1 and AtADT2, can also act as PDTs. All six AtADTs have a high sequence similarity, making it difficult to determine in silico which amino acids determine substrate specificity. Here we use domain swapping, targeted mutagenesis and pha2 yeast complementation to identify amino acids that confer PDT activity. In addition, we established a novel in vivo test of ADT activity to determine how these amino acid changes affect ADT and PDT activity of the AtADTs. Our results demonstrate that a combination of amino acids in the regulatory ACT domain are responsible for both ADT and PDT activity in the AtADTs.

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Enhanced Substrate Specificity of Thermostable Cytochrome P450 CYP175A1 by Site Saturation Mutation on Tyrosine 68

Cited by 2 publications

References 28 publications

Computationally guided bioengineering of the active site, substrate access pathway, and water channels of thermostable cytochrome P450, CYP175A1, for catalyzing the alkane hydroxylation reaction

Computationally guided bioengineering of the active site, substrate access pathway, and water channels of thermostable cytochrome P450, CYP175A1, for catalyzing the alkane hydroxylation reaction

Details matter: Defining substrate specificity in arogenate versus prephenate dehydratases

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