Acyl-ACP Substrate Recognition inBurkholderia malleiBmaI1 Acyl-Homoserine Lactone Synthase

Abstract

The acyl-homoserine lactone (AHL) autoinducer mediated quorum sensing regulates virulence in several pathogenic bacteria. The hallmark of an efficient quorum sensing system relies on the tight specificity in the signal generated by each bacterium. Since AHL signal specificity is derived from the acyl-chain of the acyl-ACP (ACP = acyl carrier protein) substrate, AHL synthase enzymes must recognize and react with the native acyl-ACP with high catalytic efficiency while keeping reaction rates with non-native acyl-ACPs low. The mechanism of acyl-ACP substrate recognition in these enzymes, however, remains elusive. In this study, we investigated differences in catalytic efficiencies for shorter and longer chain acyl-ACP substrates reacting with an octanoyl-homoserine lactone synthase Burkholderia mallei BmaI1. With the exception of two-carbon shorter hexanoyl-ACP, the catalytic efficiencies of butyryl-ACP, decanoyl-ACP, and octanoyl-CoA reacting with BmaI1 decreased by greater than 20-fold compared to the native octanoyl-ACP substrate. Furthermore, we also noticed kinetic cooperativity when BmaI1 reacted with non-native acyl-donor substrates. Our kinetic data suggest that non-native acyl-ACP substrates are unable to form a stable and productive BmaI1·acyl-ACP·SAM ternary complex and are thus effectively discriminated by the enzyme. These results offer insights into the molecular basis of substrate recognition for the BmaI1 enzyme.

A Comparative Analysis of Acyl‐Homoserine Lactone Synthase Assays

“…Indeed, we have also used this assay in the past to study BjaI and BmaI1 (an octanoyl-homoserine lactone synthase) enzymes in our laboratory. [4], [5] Based on our experience working with the DCPIP assay, we uncovered some of its facets that were not previously reported in literature. For instance, DCPIP undergoes nonspecific reduction from SAM, acyl-CoA and acyl-ACP.…”

“…Enzymes and butyryl-ACP are purified as described previously. [4], [5], [23] Graphpad Prism 6 was used to estimate kinetic constants in each assay. UV-Visible data were acquired using a Thermo Scientific Evolution 260 spectrophotometer and HPLC data were obtained using an Accela600 instrument from Thermoscientific.…”

“…[2] The N-acyl-homoserine lactone synthase enzymes in Gram-negative bacteria act as signal initiator proteins to synthesize N-acyl-homoserine lactone (AHL) autoinducers. [3–5] Depending on the acyl-substrate, AHL synthases fall into one of the two main categories: a) acyl-CoA utilizing synthases such as isovaleryl-CoA utilizing Bradyrhizobium japonicum BjaI and b) acyl-ACP utilizing synthases such as Pseudomonas aeruginosa RhlI, which uses butyryl-ACP as its acyl-substrate (Figure 1). [4] The other substrate, S-adenosyl-L-methionine (SAM), is conserved for all AHL synthases discovered so far.…”

“…Both acyl-CoA and acyl-ACP utilizing synthases use a combination of acylation and lactonization reactions to synthesize AHLs. [5] Since quorum sensing is a key step in bacterial virulence, QS inhibitors hold significant promise as antivirulent therapeutics. [6] …”

Acyl-Homoserine Lactone Based Modulators for RhlI, a Quorum Sensing Signal Synthase in Pseudomonas aeruginosa

“…21 Many AHL synthases have been shown to discriminate against non-native acyl-chain and only use a specific acyl substrate to produce enzymespecific AHL signal, thus increasing the signal to noise ratio. 22 Different receptor proteins likewise preferentially bind with their designated AHLs so that bacteria can conduct intra-specie communication without interference. Species-specific signal molecules.…”