Figure 1.
Substrates and products of acyl-HSL synthases.
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) 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.
Figure 2.
Protein phylogeny of acyl-HSL synthases from Pfam PF00765.
The sequences used in the analysis are labeled with the uniprot identifier followed by the organism identifier. BmaI1 is I1SB97_BURMA and BjaI is Q89V12_BRAJA. The clade containing CoA-utilizing acyl-HSL synthases is highlighted in red and the clades containing acyl-ACP-utilizing acyl-homoserine lactone synthases are highlighted in shades of blue. The Mig14 family (PF07395), also from the acetyltransferase-like clan (CL0257), was used as an outgroup and is collapsed as a black triangle. Labels in bold have been experimentally shown to use ACP or CoA substrates. The percentage that each branch was observed during bootstrap resampling is shown next to the branch.
Figure 3.
Structures of the acyl-substrate recognition motif.
A) Alignment of the crystal structures of LasI (1R05 in blue) [18] and EsaI (1KZF in red) [19]. 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.
Figure 4.
Protein logos of the ACP-binding loop for selected clades of acyl-HSL synthases.
The clades are identified by a characterized member. The ACP binding region is based on a previously published analysis and corresponds to amino acid residues 146–173 of LasI and 144–172 of EsaI [19]. Positively charged residues are in blue.
Table 1.
Kinetic constants for members of the acyl-HSL synthase family.
Figure 5.
Chemoenzymatic synthesis of octyl-ACP sulfide.
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 Bacillus subtilis Sfp PPant transferase (see materials and methods). B) Mass spectrum of purified octyl-ACP. The intensity is relative to the largest peak of 8960 Da. The expected mass is 8957 Da.
Figure 6.
Inhibition of acyl-HSL synthases by substrate analogs.
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.
Table 2.
Kinetics of inhibition by sulfide analogs.