ANALYSIS OF FATTY ACIDS ACTIVATION MECHANISMS IN BACTERIA: ACYL-COA AND ACYL-ACP SYNTHETASES

GALVAN VIRGINIA; GRAMAJO HUGO; ARABOLAZA ANA

Los Cocos, Córdoba

Congreso; XVII Congreso de la Sociedad Argentina de Microbiología General; 2022

Sociedad Argentina de Microbiología General

Fatty acid activation constitutes an indispensable step in numerous catabolic as well as biosynthetic pathways, producing substrates that can be oxidized, or incorporated in membrane phospholipids and in secondary metabolites. Acyl-CoA and acyl-ACP synthetases (FACS and FAAS, respectively) are the enzymes responsible of either type of activation. By obtaining the energy from ATP hydrolysis, they catalyze the synthesis of the acyl-CoA and the acyl-ACP derivative, respectively, through the formation of an acyl-adenylate intermediate. While activation by acyl-CoA formation is a highly conserved mechanism, the ability to activate fatty acids as acyl-ACP, in particular, remains exclusive to certain organisms. In many pathogenic bacteria, such as Neisseria gonorrhoeae and Chlamydia trachomatis, this capacity represents the possibility to uptake fatty acids from the host, allowing to save energy from the expensive process of biosynthesis. Several homologs of FACS/FAAS sequences exist in genomes of actinomycetes. Although, only in Mycobacterium tuberculosis the relevance of these fatty acids activation processes has been studied; where it was suggested that acyl-CoA formation is related to primary metabolism, while acyl-ACP activation represents a mechanism for connecting fatty acid synthesis with the complex PKS/NRPS systems for the production of complex lipids and secondary metabolites. As part of the adenylate forming enzymes family, both of these proteins have a group of conserved motives, and can be identified by the presence of the N-terminal specific domain PF00501. Nevertheless, until now, the preference for the acyl acceptor can only be determined through biochemical analysis. In this context, this project proposes to study this type of enzymes from a diversity of actinobacterial species with the aim of establishing a method to discriminate between both types of activation mechanisms. After carrying out homology search utilizing sequences of characterized enzymes as query, analyzing the presence of the typical PF00501 domain, performing alignments and phylogenetic tree construction, and observing the genomic contexts of these sequences, we selected a series of candidates for further experimental studies. Through heterologous expression and complementation assays in E. coli, we proposed to set up a simple method for differentiating these activities. By performing in vitro enzyme assays, we identified a new acyl-ACP synthase and its ACP partner from M. tuberculosis. Future experiments will be performed to investigate the physiological role of these proteins and to uncover their function in an uncharacterized biosynthetic pathway.