Bacillus megaterium CYP102A1 Oxidation of Acyl Homoserine Lactones and Acyl Homoserines

Abstract

Quorum sensing, the ability of bacteria to sense their own population density through the synthesis and detection of small molecule signals, has received a great deal of attention in recent years. Acyl homoserine lactones (AHLs) are a major class of quorum sensing signaling molecules. In nature, some bacteria that do not synthesize AHLs themselves have developed the ability to degrade these compounds by cleaving the amide bond or the lactone ring. By inactivating this signal used by competing bacteria, the degrading microbe is believed to gain a competitive advantage. In this work we report that CYP102A1, a widely studied cytochrome P450 from Bacillus megaterium, is capable of very efficient oxidation of AHLs and their lactonolysis products acyl homoserines. The previously known substrates for this enzyme, fatty acids, can also be formed in nature by hydrolysis of the amide of AHLs, so CYP102A1 is capable of inactivating the active parent compound and the products of both known pathways for AHL inactivation observed in nature. AHL oxidation primarily takes place at the omega-1, omega-2, and omega-3 carbons of the acyl chain, similar to this enzyme's well-known activity on fatty acids. Acyl homoserines and their lactones are better substrates for CYP102A1 than fatty acids. Bioassay of the quorum sensing activity of oxidation products reveals that the subterminally hydroxylated AHLs exhibit quorum sensing activity, but are 18-fold less active than the parent compound. In vivo, B. megaterium inactivates AHLs by a CYP102A1 dependent mechanism that must involve additional components that further sequester or metabolize the products, eliminating their quorum sensing activity. Cytochrome P450 oxidation of AHLs represents an important new mechanism of quorum quenching.

Unusual Cytochrome P450 Enzymes and Reactions

“…73). The physiological function of P450 BM-3 still remains unclear, although a role in bacterial quorum sensing mediated by oxidative inactivation of acylhomoserine lactones has been proposed (74). Similar types of P450-CPR fusion enzymes have been found in lower eukaryotes, most notably the Fusarium oxysporum membrane-associated fatty acid hydroxylase P450 foxy (CYP505) (75).…”

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…The gene responsible for this activity has yet to be identified. Two years later, the Bacillus megaterium enzyme CYP102A1, a P450 monooxygenase, was found to oxidize AHLs with both natural and nonnatural stereochemistries, as well as the various products of lactonase or acylase hydrolysis (79). A follow-up paper examining the same enzyme found that hydroxylated C12HSL retained a significant amount of its signaling activity, and B. megaterium AHL inactivation rates were only half those of analogous AiiA-expressing Bacillus spp.…”

The importance of the benzoic acid carboxylate moiety for substrate recognition by CYP199A4 from Rhodopseudomonas palustris HaA2

“…[11,28] Acyl homoserine lactones have been reported to be good substrates for CYP102A1, from Bacillus megaterium. [29] This enzyme has also been crystallised in the presence of the substrate N-palmitoyl glycine, which binds with nanomolar affinity. [30] The alcohol and amide equivalents of fatty acids are oxidised by CYP102A1 with lower activity than the fatty acids but alkanes and the methyl esters are not.…”