Fluorinated chloramphenicol acetyltransferase thermostability and activity profile: Improved thermostability by a single-isoleucine mutant

Natalya Voloshchuk; Man Xia Lee; Wan Wen Zhu; Ismet Caglar Tanrikulu; Jin Kim Montclare

doi:10.1016/j.bmcl.2007.07.107

Fluorinated chloramphenicol acetyltransferase thermostability and activity profile: Improved thermostability by a single-isoleucine mutant

Natalya Voloshchuk, Man Xia Lee, Wan Wen Zhu, Ismet Caglar Tanrikulu, Jin Kim Montclare

Chemical and Biomolecular Engineering

Research output: Contribution to journal › Article › peer-review

Abstract

A lysate-based thermostability and activity profile is described for chloramphenicol acetyltransferase (CAT) expressed in trifluoroleucine, T (CAT T). CAT and 13 single-isoleucine CAT mutants were expressed in medium supplemented with T and assayed for thermostability on cell lysates. Although fluorinated mutants, L82I T and L208I T, showed losses in thermostability, the L158I T fluorinated mutant demonstrated an enhanced thermostability relative to CAT T. Further characterization of L158I T suggested that T at position 158 contributed to a portion of the observed loss in thermostability upon global fluorination.

Original language	English (US)
Pages (from-to)	5907-5911
Number of pages	5
Journal	Bioorganic and Medicinal Chemistry Letters
Volume	17
Issue number	21
DOIs	https://doi.org/10.1016/j.bmcl.2007.07.107
State	Published - Nov 1 2007

Keywords

Mutagenesis
Non-natural amino acid
Protein engineering
Thermostability
Trifluoroleucine

ASJC Scopus subject areas

Biochemistry
Molecular Medicine
Molecular Biology
Pharmaceutical Science
Drug Discovery
Clinical Biochemistry
Organic Chemistry

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10.1016/j.bmcl.2007.07.107

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@article{d55b5d6a39cf4df8a61f00e88fb93b5d,

title = "Fluorinated chloramphenicol acetyltransferase thermostability and activity profile: Improved thermostability by a single-isoleucine mutant",

abstract = "A lysate-based thermostability and activity profile is described for chloramphenicol acetyltransferase (CAT) expressed in trifluoroleucine, T (CAT T). CAT and 13 single-isoleucine CAT mutants were expressed in medium supplemented with T and assayed for thermostability on cell lysates. Although fluorinated mutants, L82I T and L208I T, showed losses in thermostability, the L158I T fluorinated mutant demonstrated an enhanced thermostability relative to CAT T. Further characterization of L158I T suggested that T at position 158 contributed to a portion of the observed loss in thermostability upon global fluorination.",

keywords = "Mutagenesis, Non-natural amino acid, Protein engineering, Thermostability, Trifluoroleucine",

author = "Natalya Voloshchuk and Lee, {Man Xia} and Zhu, {Wan Wen} and Tanrikulu, {Ismet Caglar} and Montclare, {Jin Kim}",

note = "Funding Information: We thank the Othmer Institute, Weschler Award, NIH (GM 62523 and 5F32 GM67375-2), ACS-Fluorine Chemistry Division (MXL), and the Beckman Institute at the California Institute of Technology for financial support. We are grateful to Professors David Tirrell, Frances Arnold, Kent Kirshenbaum, and Paramjit Arora for use of equipment and reagents for this work. ",

year = "2007",

month = nov,

day = "1",

doi = "10.1016/j.bmcl.2007.07.107",

language = "English (US)",

volume = "17",

pages = "5907--5911",

journal = "Bioorganic and Medicinal Chemistry Letters",

issn = "0960-894X",

publisher = "Elsevier Ltd",

number = "21",

}

TY - JOUR

T1 - Fluorinated chloramphenicol acetyltransferase thermostability and activity profile

T2 - Improved thermostability by a single-isoleucine mutant

AU - Voloshchuk, Natalya

AU - Lee, Man Xia

AU - Zhu, Wan Wen

AU - Tanrikulu, Ismet Caglar

AU - Montclare, Jin Kim

N1 - Funding Information: We thank the Othmer Institute, Weschler Award, NIH (GM 62523 and 5F32 GM67375-2), ACS-Fluorine Chemistry Division (MXL), and the Beckman Institute at the California Institute of Technology for financial support. We are grateful to Professors David Tirrell, Frances Arnold, Kent Kirshenbaum, and Paramjit Arora for use of equipment and reagents for this work.

PY - 2007/11/1

Y1 - 2007/11/1

N2 - A lysate-based thermostability and activity profile is described for chloramphenicol acetyltransferase (CAT) expressed in trifluoroleucine, T (CAT T). CAT and 13 single-isoleucine CAT mutants were expressed in medium supplemented with T and assayed for thermostability on cell lysates. Although fluorinated mutants, L82I T and L208I T, showed losses in thermostability, the L158I T fluorinated mutant demonstrated an enhanced thermostability relative to CAT T. Further characterization of L158I T suggested that T at position 158 contributed to a portion of the observed loss in thermostability upon global fluorination.

AB - A lysate-based thermostability and activity profile is described for chloramphenicol acetyltransferase (CAT) expressed in trifluoroleucine, T (CAT T). CAT and 13 single-isoleucine CAT mutants were expressed in medium supplemented with T and assayed for thermostability on cell lysates. Although fluorinated mutants, L82I T and L208I T, showed losses in thermostability, the L158I T fluorinated mutant demonstrated an enhanced thermostability relative to CAT T. Further characterization of L158I T suggested that T at position 158 contributed to a portion of the observed loss in thermostability upon global fluorination.

KW - Mutagenesis

KW - Non-natural amino acid

KW - Protein engineering

KW - Thermostability

KW - Trifluoroleucine

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U2 - 10.1016/j.bmcl.2007.07.107

DO - 10.1016/j.bmcl.2007.07.107

M3 - Article

C2 - 17845847

AN - SCOPUS:34548862049

SN - 0960-894X

VL - 17

SP - 5907

EP - 5911

JO - Bioorganic and Medicinal Chemistry Letters

JF - Bioorganic and Medicinal Chemistry Letters

IS - 21

ER -

Fluorinated chloramphenicol acetyltransferase thermostability and activity profile: Improved thermostability by a single-isoleucine mutant

Abstract

Keywords

ASJC Scopus subject areas

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