Many prokaryotes that oxidize reduced sulfur compounds use a pathway that involves a heterodisulfide reductase-like enzyme (sHdr) system that includes lipoate binding proteins (LbpA) as essential components [1]. The LbpA proteins are not modified by the established posttranslational lipoate assembly machinery for proteins like the glycine cleavage system protein H (GcvH) [1]. Unique maturation enzymes, including a specific lipoate-protein ligase, sLpl(AB), are encoded proximate to the genes for the sHdr system. sLpl(AB) catalyzes the ATP-dependent ligation of octanoic or lipoic acid to LbpA. LipS1 and LipS2 catalyze the insertion of sulfur into bound octanyol in the absence of free lipoic acid [2].

GcvH and LbpA are similar. It is unclear how sLpl(AB) differentiates between them in organisms that contain both lipoylation substrates. We tested lipoate:protein ligases and potential substrates from five representatives of the phyla Pseudomonadota and Aquificae, with or without the capability of sulfur oxidation. Gel shift assays and MALDI-TOF mass spectrometry confirmed that the canonical E. coli Lpl(AB) lipoate:protein ligase preferentially modifies GcvH proteins, but not LbpAs. In contrast, sLpl(AB) ligases from organisms that rely on the LipS1/LipS2-dependent assembly pathway prefer LbpA proteins as substrates. GcvH proteins are usually not recognized. LbpA proteins are characterized by two conserved cysteine residues, which are absent in GcvH proteins [1]. Apo-LbpA proteins with replacements of either one or both of the cysteines to alanine still serve as substrates for sLpl(AB). This indicates that these residues are not relevant for substrate recognition. Further determinants include differentially charged protein segments, as revealed by sequence alignments. GcvH proteins feature acidic amino acid patches that are replaced by basic residues in LbpAs. The interaction between lipoate-protein ligase and its substrate appears to rely on extensive hydrogen bonding between charged amino acids and is probably dictated by surface charge complementarity.

[1] Cao et al 2018 eLife 7, e37439

[2] Neti et al. 2022 ACS Bio & Med Chem Au 2, 509