RESUMEN
Acetyl-CoA synthetase (ACS) is one of several enzymes that generate the key metabolic intermediate, acetyl-CoA. In microbes and mammals ACS activity is regulated by the post-translational acetylation of a key lysine residue. ACS in plant cells is part of a two-enzyme system that maintains acetate homeostasis, but its post-translational regulation is unknown. This study demonstrates that the plant ACS activity can be regulated by the acetylation of a specific lysine residue that is positioned in a homologous position as the microbial and mammalian ACS sequences that regulates ACS activity, occurring in the middle of a conserved motif, near the carboxyl-end of the protein. The inhibitory effect of the acetylation of residue Lys-622 of the Arabidopsis ACS was demonstrated by site-directed mutagenesis of this residue, including its genetic substitution with the non-canonical N-ε-acetyl-lysine residue. This latter modification lowered the catalytic efficiency of the enzyme by a factor of more than 500-fold. Michaelis-Menten kinetic analysis of the mutant enzyme indicates that this acetylation affects the first half-reaction of the ACS catalyzed reaction, namely, the formation of the acetyl adenylate enzyme intermediate. The post-translational acetylation of the plant ACS could affect acetate flux in the plastids and overall acetate homeostasis.
RESUMEN
The aerobic, thermophilic Actinobacterium, Thermobifida fusca has been proposed as an organism to be used for the efficient conversion of plant biomass to fatty acid-derived precursors of biofuels or biorenewable chemicals. Despite the potential of T. fusca to catabolize plant biomass, there is remarkably little data available concerning the natural ability of this organism to produce fatty acids. Therefore, we determined the fatty acids that T. fusca produces when it is grown on different carbon sources (i.e., glucose, cellobiose, cellulose and avicel) and at two different growth temperatures, namely at the optimal growth temperature of 50°C and at a suboptimal temperature of 37°C. These analyses establish that T. fusca produces a combination of linear and branched chain fatty acids (BCFAs), including iso-, anteiso-, and 10-methyl BCFAs that range between 14- and 18-carbons in length. Although different carbon sources and growth temperatures both quantitatively and qualitatively affect the fatty acid profiles produced by T. fusca, growth temperature is the greater modifier of these traits. Additionally, genome scanning enabled the identification of many of the fatty acid biosynthetic genes encoded by T. fusca.