Abstract Fatty acid synthesis in bacteria is catalyzed by a set of individual enzymes known as the type II fatty acid synthase. Acyl carrier protein (ACP) shuttles the acyl intermediates between individual pathway enzymes. In this study, we determined the solution structures of three different forms of ACP, apo‐ACP, ACP, and butyryl‐ACP under identical experimental conditions. The structural studies revealed that attachment of butyryl acyl intermediate to ACP alters the conformation of ACP. This finding supports the more general notion that the attachment of different acyl intermediates alters the ACP structure to facilitate their recognition and turnover by the appropriate target enzymes.
Staphylococcal species are a leading cause of bacterial drug-resistant infections and associated mortality. One strategy to combat bacterial drug resistance is to revisit compromised targets, and to circumvent resistance mechanisms using structure-assisted drug discovery. The folate pathway is an ideal candidate for this approach. Antifolates target an essential metabolic pathway, and the necessary detailed structural information is now available for most enzymes in this pathway. Dihydropteroate synthase (DHPS) is the target of the sulfonamide class of drugs, and its well characterized mechanism facilitates detailed analyses of how drug resistance has evolved. Here, we surveyed clinical genetic sequencing data in S. aureus to distinguish natural amino acid variations in DHPS from those that are associated with sulfonamide resistance. Five mutations were identified, F17L, S18L, T51M, E208K, and KE257_dup. Their contribution to resistance and their cost to the catalytic properties of DHPS were evaluated using a combination of biochemical, biophysical and microbiological susceptibility studies. These studies show that F17L, S18L, and T51M directly lead to sulfonamide resistance while unexpectedly increasing susceptibility to trimethoprim, which targets the downstream enzyme dihydrofolate reductase. The secondary mutations E208K and KE257_dup restore trimethoprim susceptibility closer to wild-type levels while further increasing sulfonamide resistance. Structural studies reveal that these mutations appear to selectively disfavor the binding of the sulfonamides by sterically blocking an outer ring moiety that is not present in the substrate. This emphasizes that new inhibitors must be designed that strictly stay within the substrate volume in the context of the transition state.
The transport system for pantothenic acid uptake in Escherichia coli was characterized. This transport system was specific for pantothenate, had a Kt of 0.4 microM, and had a maximum velocity of 1.6 pmol/min per 10(8) cells (45 pmol/min per mg [dry weight]). Pantothenate uptake was not reduced in osmotically shocked cells or by ATP depletion with arsenate, but was reduced greater than 90% by the dissipation of the membrane electrochemical gradient with 2,4-dinitrophenol. Sodium ions stimulated pantothenate uptake (Kt, 0.8 mM) by reducing the Kt for pantothenate by an order of magnitude. Intracellular pantothenate was rapidly phosphorylated, but phosphorylation of pantothenate was not required for uptake since pantothenate was the only labeled intracellular compound concentrated by ATP-depleted, glucose-energized cells. The data were consistent with the presence of a high-affinity pantothenate permease that concentrates the vitamin by sodium cotransport.
Staphylococcus aureus controls its membrane biophysical properties using branched-chain fatty acids (BCFAs). The branched-chain acyl-CoA precursors, utilized to initiate fatty acid synthesis, are derived from branched-chain ketoacid dehydrogenase (Bkd), a multiprotein complex that converts α-keto acids to their corresponding acyl-CoAs; however, Bkd KO strains still contain BCFAs. Here, we show that commonly used rich medias contain substantial concentrations of short-chain acids, like 2-methylbutyric and isobutyric acids, that are incorporated into membrane BCFAs. Bkd-deficient strains cannot grow in defined medium unless it is supplemented with either 2-methylbutyric or isobutyric acid. We performed a screen of candidate KO strains and identified the methylbutyryl-CoA synthetase (mbcS gene; SAUSA300_2542) as required for the incorporation of 2-methylbutyric and isobutyric acids into phosphatidylglycerol. Our mass tracing experiments show that isobutyric acid is converted to isobutyryl-CoA that flows into the even-chain acyl-acyl carrier protein intermediates in the type II fatty acid biosynthesis elongation cycle. Furthermore, purified MbcS is an ATP-dependent acyl-CoA synthetase that selectively catalyzes the activation of 2-methylbutyrate and isobutyrate. We found that butyrate and isovalerate are poor MbcS substrates and activity was not detected with acetate or short-chain dicarboxylic acids. Thus, MbcS functions to convert extracellular 2-methylbutyric and isobutyric acids to their respective acyl-CoAs that are used by 3-ketoacyl-ACP synthase III (FabH) to initiate BCFA biosynthesis.
The Fatty acid kinase (Fak) system is a two‐protein system allowing Gram‐positive pathogens to incorporate exogenous fatty acids (FA) for membrane phospholipid synthesis. Exogenous FA bind to a FA binding protein, FakB, are phosphorylated by a kinase, FakA, and used to construct the membrane. Phosphorylated FA can also be interconverted to acyl‐acyl carrier protein, via the phosphate acyltransferase PlsX, and be elongated by the fatty acid synthase type II (FASII) system. We used a combination of cellular labeling experiments, genetics, and biochemistry to characterize two FakBs of Staphylococcus aureus and three FakBs of Streptococcus pneumoniae . FakBs have structure‐specific FA binding selectivity unlike mammalian FA binding proteins. Both bacteria use one FakB (FakB1) to exclusively bind saturates and use a second FakB (FakB2) to bind monounsaturates. The additional FakB in S. pneumoniae (FakB3) binds polyunsaturates, specifically linoleate which is an abundant human FA. High‐resolution crystal structures of five FakBs show nearly identical tertiary structures allowing all FakBs to interact with other proteins involved in lipid metabolism. FakBs differ in the shape and volume of their interior FA binding pockets resulting in FA selectivity. FakB1s have a highly tailored pocket and exclude unsaturates. The FA binding pockets of FakB2s are tailored to bind monounsaturates and have a kinked tunnel to match the conformation of the cis double bond. The S. pneumoniae ‐specific FakB3 has an expanded acyl chain pocket allowing polyunsaturates to bind. FakB2s and FakB3 cannot completely exclude the binding of saturates because flexible saturated acyl chains fit into both kinked and expanded binding pockets. A lipidomic workflow was used to determine the impact of the two FakBs on the structure of the S. aureus membrane phosphatidylglycerol in a thigh infection model. Wild type S. aureus incorporated saturated and unsaturated host FA into the 1‐position of phosphatidylglycerol, whereas the fakB1 mutant was deficient in saturated FA utilization and the fakB2 mutant did not incorporate unsaturated FA. Mutagenesis, X‐ray crystallography, and NMR show that FakBs bind to anionic membranes using surface lysine residues. The membrane‐bound FA exchanges into FakB after two helices near the FA binding pocket shift outward. This mobile “flap” exposes the FA binding pocket and allows the carboxyl end of the FA to hydrogen bond to polar residues in the pocket. This work reveals the structural basis for the physiological functions of bacterial FA binding proteins in the acquisition of specific host fatty acids at the infection site. Support or Funding Information This work was supported by National Institutes of Health grant GM034496, Cancer Center Support Grant CA21765 and the American Lebanese Syrian Associated Charities. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W‐31‐109‐Eng‐38. Model for the function of FakB proteins in S. pneumoniae . Host fatty acids (FA) are incorporated into bacterial phospholipid membranes by binding to FakB. Distinct FakBs bind either saturated FA (SFA), monounsaturated FA (MUFA), or polyunsaturated FA (PUFA). FA are then phosphorylated by a kinase, FakA, and work in concert with the S. pneumoniae FASII machinery to produce substrates for acyltransferases (PlsX, PlsY) which acylate a G3P backbone and form phospholipids. Figure 1
Abstract The increasing of multidrug resistance of clinically important pathogens calls for the development of novel antibiotics with unexploited cellular targets. FA biosynthesis in bacteria is catalyzed by a group of highly conserved proteins known as the type II FA synthase (FAS II) system. Bacteria FAS II organization is distinct from its mammalian counterpart; thus the FAS II pathway offers several unique steps for selective inhibition by antibacterial agents. Some known antibiotics that target the FAS II system include triclosan, isoniazid, and thiolactomycin. Recent years have seen remarkable progress in the understanding of the genetics, biochemistry, and regulation of the FAS II system with the availability of the complete geome, sequence for many bacteria. Crystal structures of the FAS II pathway enzymes have been determined for not only the Escherichia coli model system but also other gram‐netative and gram‐positive pathogens. The protein structures have greatly facilitated structure‐based design of novel inhibitors and the improvement of existing antibacterial agents. This review discusses new developments in the discovery of inhibitors that specifically target the two reductase steps of the FAS II system, β‐ketoacyl‐acyl carrier potein (ACP) reductase and enoyl‐ACP reductase.
