Biology subjects

Sundd, M.

Publications and source records attributed to Sundd, M..

3 recordsLinked to original sources

bioRxiv2026

A thioester-thiol exchange reaction in Salmonella Invasion acyl carrier protein transfers the 4PP-bound acyl chain to a distal cysteine

Salmonella Typhimurium encodes two functionally distinct acyl carrier proteins: AcpP, involved in fatty acid synthesis, and the invasion acyl carrier protein (IacP), which mediates the acylation of SipB (Salmonella invasion protein B), a modification essential for SipB maturation and efficient host invasion. Despite growing insights, the IacP-dependent machinery governing acyl chain transfer remains largely enigmatic. Therefore, a multidisciplinary approach combining biochemistry and NMR was employed. A D35NIacP mutant (IacPN) that expresses in the apo-form was enzymatically converted into acyl-IacPN intermediates. Unexpectedly, the 1H15N HSQC spectra of the acyl-IacPN intermediates displayed two sets of resonances for several of the amides. The first was a less intense set of peaks, positioned close to the holo-peak, displaying a progressive chemical shift change with increasing acyl-chain length, reminiscent of the 4-phosphopantetheine (4-PP)-bound conformer of type II ACPs. The second, a more intense, acyl-chain-length-independent, previously uncharacterized set of peaks, was clustered near the longer 4PP-bound acyl-chain conformers (C14-). The origin of this new conformer was investigated by generating three cysteine-to-alanine mutants, C16A, C54A, and C69AIacPN. The C54AIacPN mutant displayed a single set of peaks in the acyl-IacPN intermediates, corresponding to the 4PP-bound IacPN conformer, establishing the critical role of Cys 54 in generating the uncharacterized peak and identifying it as a Cys-bound conformer. The acyl-S-carboxyamidomethyl-IacPN intermediates also displayed a single conformer, supporting the role of a cysteine in giving rise to the new peak. Collectively, our studies uncover a unique thioester-thiol exchange reaction in which the 4PP-tethered acyl chain is chemically transferred to a distal cysteine (Cys 54). Given that Cys 54 lies within the SipB interaction interface of IacP, this acyl chain relay process may contribute to IacP-mediated SipB acylation and Salmonella virulence.

biochemistry↗

bioRxiv2025

A single mutation in the DSL motif of the acyl carrier protein can prevent its in vivo modification by E. coli Holo-acyl carrier protein synthase (AcpS)

E. coli expression system is the method of choice to obtain high yields of a pure protein. However, there is always a chance that an overexpressed protein shares structural or sequence homology with the substrate of an E. coli enzyme. In such cases, the expressed protein may be partially or fully converted into the product. A notable example is the expression of acyl carrier proteins (ACP) in E. coli. Since most type II ACPs of the fatty acid synthesis pathway (FAS) have a conserved helix II, the carrier proteins are recognized as a substrate by Holo-acyl carrier protein synthase (AcpS). Thus, most ACPs express as partially or fully loaded proteins in E. coli. This undesirable modification is a concern when the objective is to obtain milligram amounts of apo-ACP. Here, using an approach combining mutagenesis, enzyme activity, and NMR, we probed for residues in ACP that can prevent this in vivo modification, without affecting Sfp (Surfactin synthetase activating enzyme) function. Taking cues from the E. coli ACP-AcpS structure (PDB 5VCB), charge neutralization mutations were designed at five different positions in EcACP that participate in ion-pair interaction with AcpS. Three of the mutants expressed solely as apo-ACP in E. coli viz. D35N, E41A and E47A/E48A. However, only the D35N mutant could be converted into holo-/acyl-ACP using Sfp in vitro, establishing mutagenesis as a viable strategy to prevent undesired modifications in vivo. As proof of principle, the mutation was applied to two unrelated ACPs that express primarily as modified proteins in E. coli -Mus musculus mitochondrial FAS ACP (MmACP) and Salmonella Typhimurium invasion acyl carrier protein (IacP). Single D35N mutation of the ACPs prevented their in vivo modification by AcpS, and the mutants were efficiently converted into holo-ACP by Sfp in vitro. These results demonstrate that D35N mutagenesis is a useful strategy to express apo-ACP in E. coli and is applicable across all type II ACPs. Furthermore, we show that holo-IacP and holo-MmACP are not recognized as substrates by AcpH (E. coli Acyl carrier protein hydrolase), and therefore they express predominantly as modified proteins in E. coli.

biochemistry↗

bioRxiv2022

Lipoate protein ligase B primarily recognizes the C8-phosphopantetheine arm of its donor substrate and weakly binds the acyl carrier protein

Lipoic acid is a sulfur containing cofactor, indispensable for the function of several metabolic enzymes. In microorganisms, lipoic acid can be salvaged from the surroundings by Lipoate protein ligase A (LplA), an ATP-dependent enzyme. Alternatively, it can be synthesized by the sequential action of Lipoate protein ligase B (LipB) and Lipoyl synthase (LipA), in a two-step reaction. LipB uptakes octanoyl-chain from C8-acyl carrier protein (C8-ACP), a byproduct of the type II fatty acid synthesis pathway, and transfers it to a conserved lysine of the lipoyl domain of a dehydrogenase. The molecular basis of substrate recognition by LipB is still not fully understood. Using E. coli LipB as a prototype, we show that the enzyme mainly recognizes the 4-phosphopantetheine tethered acyl-chain of its donor substrate, and weakly binds the apo-acyl carrier protein. It can accept octanoate-from its own ACP, noncognate ACPs, as well as C8-CoA. Further, our NMR studies demonstrate the presence of an adenine and phosphate binding site in LipB, akin to LplA. A loop containing 71RGG73 sequence, analogous to the lipoate binding loop of LplA is also conserved. Collectively, our studies highlight commonalities between LipB and LplA in their mechanism of substrate recognition. This knowledge might be of significance in the treatment of mitochondrial fatty acid synthesis related disorders.

biophysics↗