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

ABSTRACT: 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.

SUBMITTER: Broussard TC

PROVIDER: S-EPMC4813577 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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

Broussard Tyler C TC Miller Darcie J DJ Jackson Pamela P Nourse Amanda A White Stephen W SW Rock Charles O CO

The Journal of biological chemistry 20160116 12

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 d ...[more]

PMID: 26774272

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Project description:Quinolinic acid phosphoribosyltransferase (QAPRTase, EC 2.4.2.19) forms nicotinate mononucleotide (NAMN) from quinolinic acid (QA) and 5-phosphoribosyl 1-pyrophosphate (PRPP). Previously determined crystal structures of QAPRTase.QA and QAPRTase.PA.PRPP complexes show positively charged residues (Arg118, Arg152, Arg175, Lys185, and His188) lining the QA binding site. To assess the roles of these residues in the Salmonella typhimurium QAPRTase reaction, they were individually mutated to alanine and the recombinant proteins overexpressed and purified from a recombineered Escherichia coli strain that lacks the QAPRTase gene. Gel filtration indicated that the mutations did not affect the dimeric aggregation state of the enzymes. Arg175 is critical for the QAPRTase reaction, and its mutation to alanine produced an inactive enzyme. The k(cat) values for R152A and K185A were reduced by 33-fold and 625-fold, and binding affinity of PRPP and QA to the enzymes decreased. R152A and K185A mutants displayed 116-fold and 83-fold increases in activity toward the normally inactive QA analogue, nicotinic acid (NA), indicating roles for these residues in defining the substrate specificity of QAPRTase. Moreover, K185A QAPRTase displayed a 300-fold higher k(cat)/K(m) for NA over the natural substrate QA. Pre-steady-state analysis of K185A with QA revealed a burst of nucleotide formation followed by a slower steady-state rate, unlike the linear kinetics of WT. Intriguingly, pre-steady-state analysis of K185A with NA produced a rapid but linear rate for NAMN formation. The result implies a critical role for Lys185 in the chemistry of the QAPRTase intermediate. Arg118 is an essential residue that reaches across the dimer interface. Mutation of Arg118 to alanine resulted in 5000-fold decrease in k(cat) value and a decrease in the binding affinity of QA and PRPP to R152A. Equimolar mixtures of R118A with inactive or virtually inactive mutants produced approximately 50% of the enzymatic activity of WT, establishing an interfacial role for Arg118 during catalysis.

Dataset Information

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

Publications

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

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