Fig 4 - uploaded by Janusz Pętkowski
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Acetyl coenzyme A (AcCoA) as a cofactor of SACOL2570. a The structure of SACOL2570 with bound CoA. CoA binding sites (in the trimeric assembly) are formed at the interface of two monomers. b Critical residues predicted by MD simulations to participate in AcCoA binding are present in the CoA bound crystal structure of SACOL2570. All distances are shown in Å. c Both AcCoA (left panel K D = 440 μM) and CoA (right panel K D = 660 μM) bind SACOL2570
Source publication
Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of a myriad of insidious and intractable infections in humans, especially in patients with compromised immune systems and children. Here, we report the apo- and CoA-bound crystal structures of a member of the galactoside acetyltransferase superfamily from methicillin-resistant S. a...
Contexts in source publication
Context 1
... crystal structure was obtained for SACOL2570 with a bound CoA (PDB code: 3V4E). When SACOL2570 was soaked with AcCoA, only a CoA molecule was observed in the active site, likely due to hydrolysis (see below). Binding of CoA in the 3V4E crystal structure involves residues from two adjacent monomers in the homotrimeric assembly (Fig. 4a). Residues responsible for CoA binding by SACOL2570 are Asn84, Ala112*, Gly141, Lys165* and Arg182 (a star denotes residues contributed by a neighboring subunit in the trimer) (Fig. 4b). Residues responsible for the coordination of the phosphates groups of CoA are Lys165* and Arg182. The Asn84 side-chain forms a hydrogen bond ...
Context 2
... site, likely due to hydrolysis (see below). Binding of CoA in the 3V4E crystal structure involves residues from two adjacent monomers in the homotrimeric assembly (Fig. 4a). Residues responsible for CoA binding by SACOL2570 are Asn84, Ala112*, Gly141, Lys165* and Arg182 (a star denotes residues contributed by a neighboring subunit in the trimer) (Fig. 4b). Residues responsible for the coordination of the phosphates groups of CoA are Lys165* and Arg182. The Asn84 side-chain forms a hydrogen bond interaction with a water molecule in SACOL2570, but in the structure of lactose operon acetyltransferase from E. coli the corresponding residue Asn85 coordinates the acetyl group (PDB code: ...
Context 3
... demonstrated that both AcCoA and CoA bind to SACOL2570 with 1:1 stoichiometry and K D values of 460 ± 35 and 660 ± 13 μM, ΔH of -9,823 ± 200 and -8,451 ± 60 cal/mol, and ΔS of -17.6 and -13.8 cal/mol/deg, respectively (Fig. 4c). As expected the binding of AcCoA is stronger than CoA. Thus, the data confirm that SACOL2570 binds AcCoA and suggest that the protein has acetyltransferase activity. The relatively low binding affinity of SACOL2570 to AcCoA could be related to the absence of a second substrate that might impact an active site ...
Citations
... Acetyltransferase transfers the acetyl group from a wide variety of substrates including aminoglycoside antibiotics in pathogenic bacteria [68]. Galactoside O-acetyltransferase (GAT), also known as thiogalactosidetransacetylase, is one of the proteins from human pathogens that come under the National Institute for Allergy and Infectious Diseases (NIAID) Category A-C priority lists and it is mainly associated with toxin production and antibiotic resistance through detoxification process [69]. S. aureus treated with an antibiotic fusidic acid has been shown to downregulate the GAT gene [70]. ...
The present study was aimed to investigate the effect of docosanol on the protein expression profile of methicillin-resistant Staphylococcus aureus (MRSA). Thus, two-dimensional gel electrophoresis coupled with MALDI-TOF MS technique was utilized to identify the differentially regulated proteins in the presence of docosanol. A total of 947 protein spots were identified from the intracellular proteome of both control and docosanol treated samples among which 40 spots were differentially regulated with a fold change greater than 1.0. Prominently, the thiol-dependent antioxidant system and stress response proteins are downregulated in MRSA, which are critical for survival during oxidative stress. In particular, docosanol downregulated the expression of Tpx, AhpC, BshC, BrxA, and YceI with a fold change of 1.4 (p = 0.02), 1.4 (p = 0.01), 1.6 (p = 0.002), 4.9 (p = 0.02), and 1.4 (p = 0.02), respectively. In addition, docosanol reduced the expression of proteins involved in purine metabolic pathways, biofilm growth cycle, and virulence factor production. Altogether, these findings suggest that docosanol could efficiently target the antioxidant pathway by reducing the expression of bacillithiol and stress-associated proteins.
... These repeating sequence motifs were termed "isoleucine patch" or "hexapeptide repeat" and have since been found as an easily identifiable and characteristic feature of this expanding family of acyltransferases (24). AniI showed 40% sequence identity with E. coli maltose acetyltransferase (PDB: 1OCX) (23) and 36% with maltose O-acetyltransferase (PDB: 3HJJ) from Bacillus anthracis and 37% with galactoside O-acetyltransferase (PDB: 3FTT) from Staphylococcus aureus (25). Moreover, a similar "hexapeptide repeat" can be found in AniI (Fig. 2). ...
Anisomycin ( 1 ), a pyrrolidine antibiotic, exhibits diverse biological and pharmacologic activities. The biosynthetic gene cluster of 1 has been identified previously and the multistep assembly of the core benzylpyrrolidine scaffold was characterized. However, enzymatic modifications, such as acylation involved in 1 biosynthesis are unknown. In this study, the genetic manipulation of aniI proved that it encoded indispensable acetyltransferase for 1 biosynthesis. Bioinformatics analysis suggested AniI as a member of LbH-MAT-GAT sugar O-acetyltransferase, but the biochemical assay identified that its target site was the hydroxyl group of the pyrrolidine ring. AniI was found to be tolerant of acyl donors with different chain length for the biosynthesis of 1 and derivatives 12 and 13 with butyryl and isovaleryl groups, respectively. Meanwhile, it showed comparable activity towards biosynthetic intermediates and synthesized analogues, suggesting promiscuity to the pyrrolidine ring structure of 1 . These data may inspire new viable synthetic routes for the construction of more complex pyrrolidine ring scaffolds in 1 . Finally, the overexpression of aniI under the control of strong promoters contributed to the higher productivities of 1 and its analogues. These findings reported here not only improved the understanding of anisomycin biosynthesis but also expand the substrate scope of O-acetyltransferase working on the pyrrolidine ring and pave the way for future metabolic engineering construction of high-yield strain.
IMPORTANCE
Acylation is an important tailoring reaction during natural products biosynthesis. Acylation could increase the structural diversity, affect the chemical stability, volatility, biological activity and even the cellular localization of specialized compounds. Many acetyltransferases have been reported in natural product biosynthesis. The typical example of LbH-MAT-GAT sugar O-acetyltransferase subfamily was reported to catalyze the CoA-dependent acetylation of the 6-hydroxyl group of sugars. However, no protein of this family has been characterized to acetylate non-sugar secondary metabolic product. Here, AniI was found to catalyze the acylation of the hydroxyl group of the pyrrolidine ring and be tolerant of diverse acyl donors and acceptors, which made the biosynthesis more efficient and exclusive for 1 and its derivatives biosynthesis. Moreover, the overexpression of aniI serves as a successful example of genetic manipulation of a modification gene for the high production of final products and might set the stage for future metabolic engineering.
... hexapeptide repeat was an integral component of the substrate binding pockets [54]. In contrast, GCN5L1 has three XAT hexapeptide repeat motifs, with two of these regions separated by a single amino acid, and the third separated by 61 amino acids [5]. ...
General control of amino acid synthesis 5 like 1 (GCN5L1) was named due to its loose sequence alignment to GCN5, a catalytic subunit of numerous histone N-acetyltransferase complexes. Further studies show that GCN5L1 has mitochondrial and cytosolic isoforms, although functional-domain sequence alignment and experimental studies show that GCN5L1 itself does not possess intrinsic acetyltransferase activity. Nevertheless, GCN5L1 does support protein acetylation in the mitochondria and cytosol and functions as a subunit of numerous intracellular multiprotein complexes that control intracellular vacuolar organelle positioning and function. The majority of GCN5L1 studies have focused on distinct intracellular functions and in this review, we summarize these findings as well as postulate what may be common features of the diverse phenotypes linked to GCN5L1.
... Acetyltransferase can act as a detoxifying enzyme to acetylate non-metabolizing carbohydrates to retard the reentry into MRSA (Stogios et al., 2014). The acetyltransferase superfamily is demonstrated to be reduced in MRSA in the presence of fusidic acid (Luo et al., 2013). Fusidic acid also works by blocking elongation factor G on the ribosomes, thus interfering with protein synthesis (Koripella et al., 2012). ...
Methicillin-resistant Staphylococcus aureus (MRSA) is the primary microbe responsible for skin infections that are particularly difficult to eradicate. This study sought to inhibit planktonic and biofilm MRSA using furanoquinone-derived compounds containing imine moiety. A total of 19 furanoquinone analogs were designed, synthesized, and assessed for anti-MRSA potency. Among 19 compounds, (Z)-4-(hydroxyimino)naphtho[1,2-b]furan-5(4H)-one (HNF) and (Z)-4-(acetoxyimino)naphtho[1,2-b]furan-5(4H)-one (ANF) showed antibacterial activity superior to the others based on an agar diffusion assay. HNF and ANF exerted a bactericidal effect with a minimum inhibitory concentration (MIC) of 9.7 ∼ 19.5 and 2.4 ∼ 9.7 μg/ml, respectively. Both compounds were able to reduce the MRSA count by 1,000-fold in biofilm as compared to the control. In vivo efficacy was evaluated using a mouse model of skin infection. Topical application of lead compounds significantly suppressed abscess occurrence and the MRSA burden, and also ameliorated the skin-barrier function. The biochemical assay indicated the compounds' inhibition of DNA polymerase and gyrase. In silico docking revealed a favorable interaction of the compounds with DNA polymerase and gyrase although the binding was not very strong. The total DNA analysis and proteomic data suggested a greater impairment of some proteins by HNF than ANF. In general, HNF and ANF were similarly potent in MRSA inhibition in vitro and in vivo. The findings demonstrated that there was room for structural modification of furanoquinone compounds that could be used to identify anti-MRSA agent candidates.
... BtXAT has four XAT hexapeptide-repeat motifs that largely bracket its two binding domains, suggesting that these regions are either involved directly in substrate binding or aid the formation of the correct tertiary structure required for this process. A structural analysis of the SACOL2570 acetyltransferase from Staphylococcus aureus determined that the XAT hexapeptide repeat was integral to the folding of the LbH domains that form substrate-binding pockets, suggesting that the XAT hexapeptide repeat may be a necessary structure-defining feature [27]. By contrast, GCN5L1 has three XAT hexapeptide-repeat motifs, with two of these regions separated by a single amino acid and the third separated by 61 amino acids ( Figure 2A). ...
General control of amino acid synthesis 5 (GCN5) like-1 (GCN5L1) was identified as a novel gene with sequence homology to the histone acetyltransferase Gcn5. Subsequent protein-interaction studies identified GCN5L1 as a subunit of the multiprotein lysosome biogenesis complex, resulting in an alternative designation as biogenesis of lysosome-related organelle complex 1 subunit 1 (BLOS1 or BLOC1S1). Despite the distinct nomenclatures, GCN5L1/BLOS1 has been shown to play crucial roles in mitochondria, endosomes, lysosomes, and synaptic vesicle precursors (SVPs). GCN5L1/BLOS1 controls mitochondrial protein acetylation, modulates metabolic pathways, and orchestrates retrograde mitochondria-to-nucleus signaling. It also contributes to endosome-lysosome and vesicle trafficking and to endolysosomal function. Here we discuss the intracellular roles of GCN5L1/BLOS1 in the hope of linking mitochondria-centric effects to cytosolic vesicle biology.
The mycobacterial cell envelope is a defining feature of the bacteria, primarily due to its highly-ordered, relatively impermeable nature that is likely one of the key attributes that has contributed to the success of this pathogenic over the last thousand years. Peptidoglycan is a unique and essential structural element that provides much of the strength and rigidity of the mycobacterial cell envelope. Most of the enzymes involved in the biosynthetic pathway of peptidoglycan have been shown to be essential for Mycobacterium tuberculosis growth. Mycobacterium tuberculosis GlmU is an essential bifunctional N-acetyltransferase, uridylyltransferase enzyme involved in the formation of uridine-diphosphate N-acetylglucosamine, which is the universal donor of N-acetylglucosamine for both peptidoglycan and lipopolysaccharide biosynthesis. This enzyme catalyses acetylation of glucosamine 1-phosphate, followed by uridylylation of N-acetylglucosamine 1-phosphate. Detailed characterisation of the kinetic mechanism ascertained that acetyl transfer progresses by the formation of a ternary complex, with acetyl coenzyme A binding preceding glucosamine 1-phosphate and coenzyme A the last product to dissociate. A novel ternary complex crystal structure, with glucose 1-phosphate and acetyl-coenzyme A, identified a candidate general base involved in the deprotonation of glucosamine 1-phosphate, as well as other important active site residues for substrate binding and catalysis. pH-rate studies and site-directed mutagenesis led to assignment of Histidine 374 as the catalytic general base. Solvent kinetic isotope effect experiments and pH-rate studies identified that acetyl transfer is partially rate-limiting. Small molecule screening led to the identification of novel inhibitors of GlmU catalysed acetyl transfer reaction. Inhibitors identified from the GSK TB set, were confirmed as GlmU interacting compounds and shown to inhibit the growth of M. tuberculosis. These results show that knowledge of the kinetic and catalytic mechanism enabled optimisation of a thorough screening approach that identified novel inhibitors that demonstrated that GlmU acetyltransferase activity is essential for M. tuberculosis growth.
