Divalent cation biding to reduced and octanoyl acyl‐carrier protein

Abstract

Mn(I1) EPR binding studies with reduced acyl-carrier protein (ACP-SH) strongly suggest the presence of two relatively high-affinity manganese-binding sites (average &/site z 80 pM) at physiological pH. Lowering the pH or titrating with sodium chloride reduces the average number of bound divalent cations and decreases the binding affinity. This is consistent with the idea that anionic ligand(s), e. g. the carboxylate of glutamic or aspartic acid, on the protein are involved in manganese ion coordination. At pH values above 8.0, binding affinity is also reduced, whereas the average number of bound metal ions increases to about five at pH 8.5. By interacting weakly with divalent cations (average &/site z 1 mM), octanoyl acyl-carrier protein (OcoACP) exhibits dramatically different metal-ion-binding properties compared to ACP-SH. Calcium and magnesium can compete in either ACP species for manganese binding. Photochemically-induced dynamic nuclear polarisation 'H-NMR experiments strongly suggest that ACP-SH and OcoACP undergo at pH-induced conformational change between pH 5.5 and pH 7.0, and that divalent cations stabilize the protein against such pH-induced structural perturbations.Fatty acid biosynthesis is a central biochemical pathway involving several enzymatic steps [I -31. Acyl-carrier proteins (ACP) are coenzymes which function in acyl-group-transfer reactions, chain-elongation reactions by successive addition of two-carbon fragments from malonyl-CoA, and as anchors for reduction and dehydration reactions that ultimately lead to a saturated fatty acid [2]. Since ACP is active in a variety of differing acylated states involved in various enzymatic reactions and, therefore, with a number of different enzymes, it is likely that ACP has the ability to modify or fine-tune its conformation to interact efficiently in all situations and, perhaps, even effect regulation of fatty acid biosynthesis.The ability to isolate ACP from Escherichiu coli as a soluble protein, and not as part of a tightly associated protein complex found in higher organisms, has made it the most studied among all ACPs which, in any event, possess a high degree of sequence similarity [2]. Of the 77 amino acids (8847 Da) found in E. coli ACP, 22 are acidic (glutamic and aspartic acid residues) and six are basic (lysine, arginine and histidine residues) [4, 51. This gives the protein an net charge of -16. To date, a secondary backbone structure for E. coli ACP-SH (reduced) has been derived from two-dimensional 'H-NMR studies [6]. The following segments are proposed to be helical: residues 3 -15,37 -51,56 -63 and 65 -75; these are connected by extended chain and p-turn structure. The three-dimensional structure of ACP-SH, determined by NMR pseudo-energy and distance geometry calculations [7], have identified three helical regions: residues 3-14, 37-51 and 65-75, which run anti-parallel to each other. Octanoyl-ACP, an S-acylated species of ACP, shows a similar overall conformation with Correspondence to K .

NMR Solution Structure and Biophysical Characterization of Vibrio harveyi Acyl Carrier Protein A75H

“…2B). This stacked conformation is also observed in the EcACP structure (6) and positions the residues optimally for cation-pi interactions (33) and is consistent with the observation that His-75 in EcACP has an elevated pK a between 7.5 and 8.0 (34). Fig.…”

Steady-State Kinetics and Mechanism of LpxD, the N-Acyltransferase of Lipid A Biosynthesis

“…6A) and K + ions (not shown) do not inhibit LpxD activity. These findings are consistent with reports that ACP has a high affinity for both Ca 2+ and Mg 2+ , but only a weak affinity for Na + and K + (37,38). Fig.…”

Proteome Analysis of B. subtilis in Response to Calcium

“…These findings are supported by previous reports, which showed that the E. coli ACP, and more recently Vibrio harvey ACP bind Ca 2+ with high affinity [54][55][56]. However, the role of Ca 2+ and ACP has not been analyzed in vivo.…”