A Complex of LaoA and LaoB Acts as a Tat-Dependent Dehydrogenase for Long-Chain Alcohols in Pseudomonas aeruginosa

Abstract

The opportunistic pathogen Pseudomonas aeruginosa can utilize unusual carbon sources like sodium dodecyl sulfate (SDS) and alkanes. Whereas the initiating enzymatic steps of the corresponding degradation pathways have been characterized in detail, the oxidation of the emerging long-chain alcohols found little attention. Recently, the genes for the Lao ( l ong-chain a lcohol/ a ldehyde o xidation) system were discovered to be involved in the oxidation of long-chain alcohols derived from SDS and alkane degradation. In the Lao-system, LaoA is predicted to contain the catalytic site, however, according to genetic studies, efficient long-chain alcohol oxidation additionally required the Tat-dependent protein LaoB. In the present study, the Lao-system was further characterized. In vivo analysis revealed that the Lao-system complements the substrate spectrum of the well-described Exa-system, which is required for growth with ethanol and other short-chain alcohols. Mutational analysis revealed that the Tat-site of LaoB was required for long-chain alcohol oxidation activity strongly suggesting a periplasmic localization of the process. Purified LaoA was only fully active when co-purified with LaoB. Interestingly, in vitro activity of the purified LaoAB-complex also depended on the presence of the Tat-site. The co-purified LaoAB-complex contained a flavin co-factor and preferentially oxidized a range of saturated, unbranched primary alcohols. Furthermore, the LaoAB-complex could reduce cytochrome c 550 type redox carriers like ExaB, a subunit of the Exa alcohol dehydrogenase system. LaoAB-complex activity was stimulated by rhamnolipids in vitro . In summary, LaoAB constitutes an unprecedented protein complex with specific properties apparently required for oxidizing long-chain alcohols. IMPORTANCE Pseudomonas aeruginosa is a major threat to public health. Its ability to thrive in clinical settings, water distribution systems or even jet fuel tanks is linked to detoxification and degradation of diverse hydrophobic substrates that are metabolized via alcohol intermediates. Our study illustrates a novel flavoprotein long-chain alcohol dehydrogenase consisting of a facultative two-subunit complex, which is unique among related enzymes while the homologs of the corresponding genes are found in numerous bacterial genomes. Understanding the involved catalytic and compartmentalization processes is of great interest for biotechnological and hygiene research as it may be a potential starting point for rationally designing novel antibacterial substances with high specificity against this opportunistic pathogen.

A Plasmid-Borne Gene Cluster Flanked by Two Restriction-Modification Systems Enables an Arctic Strain of Psychrobacter sp. to Decompose SDS

“…(e.g., [ 27 ]). Recently, the LaoABCR system for the oxidation of the products of SDS hydrolysis in Pseudomonas aeruginosa PAO1 was characterized [ 28 , 29 , 30 ]. In this case, both positive and negative regulation mechanisms were shown to control the studied metabolic pathway, with, i.a., SDS and long-chain acyl-CoA esters acting as inducers of gene expression in a step-wise manner [ 29 ].…”