Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family

Broussard, Tyler C.; Miller, Darcie J.; Jackson, Pamela; Nourse, Amanda; White, Stephen W.; Rock, Charles O.

doi:10.1074/jbc.M115.706820

Title: Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family

Journal Article · Sat Jan 16 00:00:00 EST 2016 · Journal of Biological Chemistry

DOI:https://doi.org/10.1074/jbc.M115.706820· OSTI ID:1243114

Broussard, Tyler C. ^[1]; Miller, Darcie J. ^[1]; Jackson, Pamela ^[1]; Nourse, Amanda ^[1]; White, Stephen W. ^[1]; Rock, Charles O. ^[1]

St. Jude Children's Hospital, Memphis, TN (United States)

Fatty acid kinase (Fak) is a ubiquitous Gram-positive bacterial enzyme consisting of an ATP-binding protein (FakA) that phosphorylates the fatty acid bound to FakB. In Staphylococcus aureus, Fak is a global regulator of virulence factor transcription and is essential for the activation of exogenous fatty acids for incorporation into phospholipids. The 1.2-Å x-ray structure of S. aureus FakB2, activity assays, solution studies, site-directed mutagenesis, and in vivo complementation were used to define the functions of the five conserved residues that define the FakB protein family (Pfam02645). The fatty acid tail is buried within the protein, and the exposed carboxyl group is bound by a Ser-93-fatty acid carboxyl-Thr-61-His-266 hydrogen bond network. The guanidinium of the invariant Arg-170 is positioned to potentially interact with a bound acylphosphate. The reduced thermal denaturation temperatures of the T61A, S93A, and H266A FakB2 mutants illustrate the importance of the hydrogen bond network in protein stability. The FakB2 T61A, S93A, and H266A mutants are 1000-fold less active in the Fak assay, and the R170A mutant is completely inactive. All FakB2 mutants form FakA(FakB2)2 complexes except FakB2(R202A), which is deficient in FakA binding. Allelic replacement shows that strains expressing FakB2 mutants are defective in fatty acid incorporation into phospholipids and virulence gene transcription. These conserved residues are likely to perform the same critical functions in all bacterial fatty acid-binding proteins.

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Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States)

Sponsoring Organization:: National Inst. of Health; Cancer Center Support; NIGMS; NIAID; American Lebanese Syrian Associated Charities

Grant/Contract Number:: GM034496; T32 AI106700; CA21765

OSTI ID:: 1243114

Journal Information:: Journal of Biological Chemistry, Vol. 291, Issue 12; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

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Cited by: 18 works

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crystal structure
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site-directed mutagenesis
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Title: Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family

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