Insights into the Biosynthesis of Dehydroalanines in Goadsporin

Abstract

Dehydroalanines in goadsporin are proposed to be formed by GodF and GodG, which show slight homology to the N-terminal glutamylation and C-terminal elimination domains, respectively, of LanB, a class I lanthipeptide dehydratase. Although similar, separated-type LanBs are conserved among thiopeptides and indispensable for their biosynthesis and biological activities, these enzymes had not yet been characterized. Here, we identified goadsporin B, which has unmodified Ser4 and Ser14, from both godF and godG disruptants. The godG disruptant also produced goadsporin C, a glutamylated-Ser4 variant of goadsporin B. These results suggested that dehydroalanines are formed by glutamylation and glutamate elimination. NMR analysis revealed for the first time that the glutamyl group was attached to a serine via an ester bond, by the catalysis of LanB-type enzymes. Our findings provide insights into the function of separated-type LanBs involved in the biosynthesis of goadsporin and thiopeptides.

Capture of micrococcin biosynthetic intermediates reveals C-terminal processing as an obligatory step for in vivo maturation

“…S5). A similar pattern of multiple glutamylations has previously been observed for nonthiopeptide RiPP pathways (22,25); however, in the in vitro reconstituted thiomuracin system, only a monoglutamylated intermediate accumulates when the TclL ortholog (TbtC) is omitted (11). This discrepancy may be due to the different reaction conditions (in vitro versus in vivo) or to an intrinsic difference in how this two-step dehydration process is coordinated in the two biosynthetic systems.…”

Mechanistic Understanding of Lanthipeptide Biosynthetic Enzymes

“…Chemoselective activation by glutamate via its α-carboxylate is illustrated here as has been demonstrated for the nonlanthipeptide RiPP goadsporin. 195 Conserved LanB residues predicted by mutagenesis studies on NisB to be important for transesterification and elimination are shown in gray. The exact roles of these residues remain to be established.…”

YcaO-Dependent Posttranslational Amide Activation: Biosynthesis, Structure, and Function

“…202, 203 Genetic deletion studies support a biosynthetic pathway in which the azoles are formed first by a fused cyclodehydratase (GodD) and a dehydrogenase (GodE). 204 In the next likely step, dehydroamino acids are formed by what is now termed a split LanB dehydratase that catalyzes the glutamylation of select Ser and Thr (GodG) and subsequent elimination (GodF) to yield the corresponding dehydroalanine and dehydrobutyrine moieties. 204, 205 Putative membrane-associated proteases (GodB and GodC) cleave the modified precursor peptide (GodA), and an N-acetyltransferase (GodH) modifies the newly formed N-terminus.…”

“…204 In the next likely step, dehydroamino acids are formed by what is now termed a split LanB dehydratase that catalyzes the glutamylation of select Ser and Thr (GodG) and subsequent elimination (GodF) to yield the corresponding dehydroalanine and dehydrobutyrine moieties. 204, 205 Putative membrane-associated proteases (GodB and GodC) cleave the modified precursor peptide (GodA), and an N-acetyltransferase (GodH) modifies the newly formed N-terminus. 206, 207 Transcription of the goadsporin BGC is controlled by GodR, an activator, 208 whose overexpression results in increased production of goadsporin, although the best titers were achieved through heterologous expression in S. lividans and co-culturing with Tsukamurella pulmonis .…”

“…45, 205, 269 A formal [4+2] cycloaddition of two dehydroalanines forms the six-membered nitrogenous heterocycle (Figure 13). 45, 270 The identity of the [4+2] cycloaddition enzyme was inferred by its absence in goadsporin (section 2.7), 204, 213 which structurally resembles a presumed intermediate during thiopeptide biosynthesis. Deleting this gene in thiopeptide BGCs resulted in the isolation of linear intermediates lacking this 6-membered heterocycle.…”