Structural analysis and interaction studies of acyl-carrier protein (acpP) of Staphylococcus aureus, an extraordinarily thermally stable protein

Abstract

Enzymol. 459, 395-433; Halavaty, A.S., Kim, Y., Minasov, G., Shuvalova, L., Dubrovska, I., Winsor, J., Zhou, M., Onopriyenko, O., Skarina, T., Papazisi, L., et al. (2012). Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria. Acta Crystallogr. Sect. D Biol. Crystallogr. 68, 1359-1370]. The inactive apo-form is converted to the active holo-enzyme by acyl-carrier protein synthase (acpS) through addition of a 4'-phosphopantetheine group from coenzyme A to a conserved serine residue of acpP [Flugel, R.S., Hwangbo, Y., Lambalot, R.H., Cronan, J.E., and Walsh, C.T. (2000). Holo-(acyl-carrier protein) synthase and phosphopantetheinyl transfer in Escherichia coli. J. Biol. Chem. 275, 959-968; Lambalot, R.H. and Walsh, C.T. (1995). Cloning, overproduction, and characterization of the Escherichia coli holo-acyl-carrier protein synthase. J. Biol. Chem. 270, 24658-24661]. Once activated, acpP acts as an anchor for the growing fatty acid chain. Structural data from X-ray crystallographic analysis reveals that, despite its small size (8 kDa), acpP adopts a distinct, mostly α-helical structure when complexed with acpS [Halavaty, A.S., Kim, Y., Minasov, G., Shuvalova, L., Dubrovska, I., Winsor, J., Zhou, M., Onopriyenko, O., Skarina, T., Papazisi, L., et al. (2012). Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria. Acta Crystallogr. Sect. D Biol. Crystallogr. 68, 1359-1370; Byers, D.M. and Gong, H. (2007). Acyl carrier protein: structure-function relationships in a conserved multifunctional protein family. Biochem. Cell Biol. 85, 649-662]. We expressed and purified recombinant, active S. aureus acpP from Escherichia coli and mimicked the beginning of fatty acid biosynthesis by employing an [14C]-acp loading assay. Surprisingly, acpP remained functional even after heat treatment at 95°C for up to 10 min. NMR data from 2D-HSQC experiments as well as interaction studies with acpS confirmed that acpP is structured and active both before and after heat treatment, with no significant differences between the two. Thus, our data suggest that S. aureus acpP is a highly stable protein capable of maintaining its structure at high temperatures.

Metabolic network model guided engineering ethylmalonyl-CoA pathway to improve ascomycin production in Streptomyces hygroscopicus var. ascomyceticus

“…In transaldolase-overexpressing strain, the carbon flux to E4P was enhanced which resulted in an increased supply of shikimate acid [ 38 ]. The targets MOHMT, PANTS, PNTK, PPNCL2, DPR and PPCDC are involved in the synthesis of pantetheine 4′-phosphate (an intermediate in coenzyme A biosynthesis pathway), which is an essential prosthetic group of acyl carrier protein (ACP), and a key cofactor in the biosynthesis of polyketides and fatty acids [ 51 – 53 ]. The next target, CCCR ( f PH = 1.906) was catalyzed by crotonyl-CoA carboxylase/reductase (Ccr).…”

The influence of commonly used tags on structural propensities and internal dynamics of peptides

“…The peptides investigated are part of a larger quest for antibacterial activity [31], they are: A0 having the sequence RKKRLKLLKRLL, with flA0 bearing a fluorescein tag (with an Ahx spacer) at the N-terminus; S2 having the amino acid sequence RIQALYSKQPKLVERILTKNEQ; and P1 having the sequence DSLDIAELVMELEDEFGTEIPD, with bioS2 and bioP1 respectively being biotinylated at the N-terminus.…”