Molecular Basis for the Substrate Specificity of Quorum Signal Synthases

In several Proteobacteria, LuxI-type enzymes catalyze the biosynthesis of acyl–homoserine lactones (AHL) signals using S-adenosyl– L-methionine and either cellular acyl carrier protein (ACP)-coupled fatty acids or CoA–aryl/acyl moieties as progenitors. Little is known about the molecular mechanism of signal biosynthesis, the basis for substrate specificity, or the rationale for donor specificity for any LuxI member. Here, we present several cocrystal structures of BjaI, a CoAdependent LuxI homolog that represent views of enzyme complexes that exist along the reaction coordinate of signal synthesis. Complementary biophysical, structure–function, and kinetic analysis define the features that facilitate the unusual acyl conjugation with S-adenosylmethionine (SAM). We also identify the determinant that establishes specificity for the acyl donor and identify residues that are critical for acyl/aryl specificity. These results highlight howa prevalent scaffold has evolved to catalyze quorum signal synthesis and provide a framework for the design of small-molecule antagonists of quorum signaling

Enzymatic diversity in lipoic acid modification of proteins

Escherichia coli is one of a few organisms with a well characterized and complete metabolic model for lipoic acid metabolism. In this thesis I make discoveries through analysis of the diversity of lipoic acid metabolic genes in other organisms. By examining the distribution and phylogeny of the lipoate ligase protein family, I and others have found gaps and inconsistencies in our knowledge of lipoic acid metabolic pathways. I examine select proteins through heterologous expression in E. coli, allowing complementation studies and biochemical analysis. This enables prediction of lipoic acid metabolism in various organisms and reveals novel pathways and enzymology

Evolution of acyl-substrate recognition by a family of acyl-homoserine lactone synthases.

Members of the LuxI protein family catalyze synthesis of acyl-homoserine lactone (acyl-HSL) quorum sensing signals from S-adenosyl-L-methionine and an acyl thioester. Some LuxI family members prefer acyl-CoA, and others prefer acyl-acyl carrier protein (ACP) as the acyl-thioester substrate. We sought to understand the evolutionary history and mechanisms mediating this substrate preference. Our phylogenetic and motif analysis of the LuxI acyl-HSL synthase family indicates that the acyl-CoA-utilizing enzymes evolved from an acyl-ACP-utilizing ancestor. To further understand how acyl-ACPs and acyl-CoAs are recognized by acyl-HSL synthases we studied BmaI1, an octanoyl-ACP-dependent LuxI family member from Burkholderia mallei, and BjaI, an isovaleryl-CoA-dependent LuxI family member from Bradyrhizobium japonicum. We synthesized thioether analogs of their thioester acyl-substrates to probe recognition of the acyl-phosphopantetheine moiety common to both acyl-ACP and acyl-CoA substrates. The kinetics of catalysis and inhibition of these enzymes indicate that they recognize the acyl-phosphopantetheine moiety and they recognize non-preferred substrates with this moiety. We find that CoA substrate utilization arose through exaptation of acyl-phosphopantetheine recognition in this enzyme family

Identification of a Biosynthetic Gene Cluster and the Six Associated Lipopeptides Involved in Swarming Motility of Pseudomonas syringae pv. tomato DC3000▿ †

Pseudomonas species are known to be prolific producers of secondary metabolites that are synthesized wholly or in part by nonribosomal peptide synthetases. In an effort to identify additional nonribosomal peptides produced by these bacteria, a bioinformatics approach was used to “mine” the genome of Pseudomonas syringae pv. tomato DC3000 for the metabolic potential to biosynthesize previously unknown nonribosomal peptides. Herein we describe the identification of a nonribosomal peptide biosynthetic gene cluster that codes for proteins involved in the production of six structurally related linear lipopeptides. Structures for each of these lipopeptides were proposed based on amino acid analysis and mass spectrometry analyses. Mutations in this cluster resulted in the loss of swarming motility of P. syringae pv. tomato DC3000 on medium containing a low percentage of agar. This phenotype is consistent with the loss of the ability to produce a lipopeptide that functions as a biosurfactant. This work gives additional evidence that mining the genomes of microorganisms followed by metabolite and phenotypic analyses leads to the identification of previously unknown secondary metabolites

Chemoenzymatic synthesis of octyl-ACP sulfide.

<p>A) Synthesis of octyl ACP. In this two-step reaction, octyl-CoA sulfide was first synthesized by coupling octyl bromide with Coenzyme A, followed by enzymatic transfer of the alkyl-PPant to apo-ACP using <i>Bacillus subtilis</i> Sfp PPant transferase (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112464#s4" target="_blank">materials and methods</a>). B) Mass spectrum of purified octyl-ACP. The intensity is relative to the largest peak of 8960 Da. The expected mass is 8957 Da.</p

Substrates and products of acyl-HSL synthases.

<p>A) Acyl-HSL synthases have two substrates and three products. The substrate acyl group is attached as a thioester to an acyl carrier: either an acyl carrier protein or coenzyme A<b>.</b> B) Comparison of the structures of acyl-ACP and acyl-CoA. Both carriers have an acyl-phosphopantetheine (acyl-PPant) moiety. Thioether analogs of these thioester substrates lack the acyl oxygen.</p

Inhibition of acyl-HSL synthases by substrate analogs.

<p>The best-fit models of inhibition are graphed. The µM concentration of inhibitor for each experiment is shown next to the curve. A) Substrate-velocity curves of mixed inhibition of 0.4 µM BmaI1 by octyl-ACP. B) Substrate-velocity curves of competitive inhibition of 0.5 µM BjaI with varying isopentyl-CoA.</p

Structures of the acyl-substrate recognition motif.

<p>A) Alignment of the crystal structures of LasI (1R05 in blue) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112464#pone.0112464-Watson1" target="_blank">[18]</a> and EsaI (1KZF in red) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112464#pone.0112464-Gould1" target="_blank">[19]</a>. The two structures have a root-mean-square deviation of 1.45 Å for 124 amino acid α carbons. The conserved α-helix proposed to interact with ACP is circled in yellow. The active site cleft is behind this helix next to the conserved β-sheet. B) The LasI structure rotated 90° about the Z axis with positively-charged residues in the motif displayed.</p