The Central Andes region displays unexplored ecosystems of shallow lakes and salt flats at mean altitudes of 3,700 m. Being isolated and hostile, these so-called “High-Altitude Andean Lakes” (HAAL) are pristine and have been exposed to little human influence. HAAL proved to be a rich source of microbes showing interesting adaptations to life in extreme settings (poly-extremophiles) such as alkalinity, high concentrations of arsenic and dissolved salts, intense dryness, large daily ambient thermal amplitude, and extreme solar radiation levels. This work reviews HAAL microbiodiversity, taking into account different microbial niches, such as plankton, benthos, microbial mats and microbialites. The modern stromatolites and other microbialites discovered recently at HAAL are highlighted, as they provide unique modern -though quite imperfect- analogues of environments proxy for an earlier time in Earth’s history (volcanic setting and profuse hydrothermal activity, low atmospheric O2 pressure, thin ozone layer and high UV exposure). Likewise, we stress the importance of HAAL microbes as model poly-extremophiles in the study of the molecular mechanisms underlying their resistance ability against UV and toxic or deleterious chemicals using genome mining and functional genomics. In future research directions, it will be necessary to exploit the full potential of HAAL poly-extremophiles in terms of their biotechnological applications. Current projects heading this way have yielded detailed molecular information and functional proof on novel extremoenzymes: i.e. DNA repair enzymes and arsenic efflux pumps for which medical and bioremediation applications, respectively, are envisaged. But still, much effort is required to unravel novel functions for this and other molecules that dwell in a unique biological treasure despite its being hidden high up, in the remote Andes.

Mycolic acids and multimethyl-branched fatty acids are found uniquely in the cell envelope of pathogenic mycobacteria. These unusually long fatty acids are essential for the survival, virulence, and antibiotic resistance of Mycobacterium tuberculosis . Acyl-CoA carboxylases (ACCases) commit acyl-CoAs to the biosynthesis of these unique fatty acids. Unlike other organisms such as Escherichia coli or humans that have only one or two ACCases, M. tuberculosis contains six ACCase carboxyltransferase domains, AccD1–6, whose specific roles in the pathogen are not well defined. Previous studies indicate that AccD4, AccD5, and AccD6 are important for cell envelope lipid biosynthesis and that its disruption leads to pathogen death. We have determined the 2.9-Å crystal structure of AccD5, whose sequence, structure, and active site are highly conserved with respect to the carboxyltransferase domain of the Streptomyces coelicolor propionyl-CoA carboxylase. Contrary to the previous proposal that AccD4–5 accept long-chain acyl-CoAs as their substrates, both crystal structure and kinetic assay indicate that AccD5 prefers propionyl-CoA as its substrate and produces methylmalonyl-CoA, the substrate for the biosyntheses of multimethyl-branched fatty acids such as mycocerosic, phthioceranic, hydroxyphthioceranic, mycosanoic, and mycolipenic acids. Extensive in silico screening of National Cancer Institute compounds and the University of California, Irvine, ChemDB database resulted in the identification of one inhibitor with a K _i of 13.1 μM. Our results pave the way toward understanding the biological roles of key ACCases that commit acyl-CoAs to the biosynthesis of cell envelope fatty acids, in addition to providing a target for structure-based development of antituberculosis therapeutics.

Pathogenic mycobacteria contain a variety of unique fatty acids that have methyl branches at an even-numbered position at the carboxyl end and a long n -aliphatic chain. One such group of acids, called mycocerosic acids, is found uniquely in the cell wall of pathogenic mycobacteria, and their biosynthesis is essential for growth and pathogenesis. Therefore, the biosynthetic pathway of the unique precursor of such lipids, methylmalonyl coenzyme A (CoA), represents an attractive target for developing new antituberculous drugs. Heterologous protein expression and purification of the individual subunits allowed the successful reconstitution of an essential acyl-CoA carboxylase from Mycobacterium tuberculosis , whose main role appears to be the synthesis of methylmalonyl-CoA. The enzyme complex was reconstituted from the α biotinylated subunit AccA3, the carboxyltransferase β subunit AccD5, and the ε subunit AccE5 (Rv3281). The kinetic properties of this enzyme showed a clear substrate preference for propionyl-CoA compared with acetyl-CoA (specificity constant fivefold higher), indicating that the main physiological role of this enzyme complex is to generate methylmalonyl-CoA for the biosynthesis of branched-chain fatty acids. The α and β subunits are capable of forming a stable α6-β6 subcomplex but with very low specific activity. The addition of the ε subunit, which binds tightly to the α-β subcomplex, is essential for gaining maximal enzyme activity.

Two acyl-CoA carboxylases fromStreptomyces coelicolor have been successfully reconstituted from their purified components. Both complexes shared the same biotinylated α subunit, AccA2. The β and the ε subunits were specific from each of the complexes; thus, for the propionyl-CoA carboxylase complex the β and ε components are PccB and PccE, whereas for the acetyl-CoA carboxylase complex the components are AccB and AccE. The two complexes showed very low activity in the absence of the corresponding ε subunits; addition of PccE or AccE dramatically increased the specific activity of the enzymes. The kinetic properties of the two acyl-CoA carboxylases showed a clear difference in their substrate specificity. The acetyl-CoA carboxylase was able to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity. The propionyl-CoA carboxylase could not recognize acetyl-CoA as a substrate, whereas the specificity constant for propionyl-CoA was 2-fold higher than for butyryl-CoA. For both enzymes the ε subunits were found to specifically interact with their carboxyltransferase component forming a β-ε subcomplex; this appears to facilitate the further interaction of these subunits with the α component. The ε subunit has been found genetically linked to several carboxyltransferases of differentStreptomyces species; we propose that this subunit reflects a distinctive characteristic of a new group of acyl-CoA carboxylases.