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

Kinetic constants for members of the acyl-HSL synthase family.

a<p>RhlI kinetic constants are from another study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112464#pone.0112464-Raychaudhuri1" target="_blank">[33]</a>.</p>b<p><i>k</i>_cat/<i>K</i>_m ratio = (<i>k</i>_cat/<i>K</i>_m)^{preferred substrate}/(<i>k</i>_cat/<i>K</i>_m)^{non-preferred substrate}.</p><p>Kinetic constants for members of the acyl-HSL synthase family.</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

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

Kinetics of inhibition by sulfide analogs.

<p>Kinetics of inhibition by sulfide analogs.</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