Search

Acylation of lysine is an important protein modification regulating diverse biological processes. It was recently demonstrated that members of the human Sirtuin family are capable of catalyzing long chain deacylation, in addition to the well-known NAD+-dependent deacetylation activity [Feldman, J. L., Baeza, J., and Denu, J. M. (2013) J. Biol. Chem. 288, 31350–31356]. Here we provide a detailed kinetic and structural analysis that describes the interdependence of NAD+-binding and acyl-group selectivity for a diverse series of human Sirtuins, SIRT1–SIRT3 and SIRT6. Steady-state and rapid-quench kinetic analyses indicated that differences in NAD+ saturation and susceptibility to nicotinamide inhibition reflect unique kinetic behavior displayed by each Sirtuin and depend on acyl substrate chain length. Though the rate of nucleophilic attack of the 2'-hydroxyl on the C1'-O-alkylimidate intermediate varies with acyl substrate chain length, this step remains rate-determining for SIRT2 and SIRT3; however, for SIRT6, this step is no longer rate-limiting for long chain substrates. Cocrystallization of SIRT2 with myristoylated peptide and NAD+ yielded a co-complex structure with reaction product 2'-O-myristoyl-ADP-ribose, revealing a latent hydrophobic cavity to accommodate the long chain acyl group, and suggesting a general mechanism for long chain deacylation. Comparing two separately determined co-complex structures containing either a myristoylated peptide or 2'-O-myristoyl-ADP-ribose indicates there are conformational changes at the myristoyl–ribose linkage with minimal structural differences in the enzyme active site. During the deacylation reaction, the fatty acyl group is held in a relatively fixed position. We describe a kinetic and structural model to explain how various Sirtuins display unique acyl substrate preferences and how different reaction kinetics influence NAD+ dependence. The biological implications are discussed. [ABSTRACT FROM AUTHOR]

Ghrelin O-acyltransferase (GOAT) is an integral membrane acyltransferase responsible for catalyzing a serine-octanoylation posttranslational modification within the peptide hormone ghrelin. Ghrelin requires this octanoylation for its biological activity in stimulating appetite and in regulating other physiological pathways involved in energy balance. Blocking ghrelin acylation using GOAT inhibitors is a new potential avenue to treat health conditions impacted by ghrelin signaling, such as obesity and diabetes. Designing novel and potent GOAT inhibitors as potential therapeutics requires insight into the interactions between the ghrelin and octanoyl coenzyme A substrates and the GOAT active site. Through structure-activity investigation of ghrelin-mimetic peptide substrates and inhibitors, we have analyzed the amino acid selectivity of the enzyme as well as the functional groups involved in substrate recognition by human GOAT (hGOAT). This analysis reveals that hGOAT both prefers and tolerates a distinct set of chemical properties at each position within the N-terminal sequence of ghrelin and that sequence elements downstream of the ghrelin N-terminal sequence contribute to ghrelin binding to hGOAT. We also found that the hGOAT active site exhibits a marked preference for binding an eight-carbon acyl chain, which potentially explains the biological observation of ghrelin octanoylation in light of the acyl donor promiscuity reported for GOAT. Bioinformatics analysis, guided by our reactivity data, supports the conclusion that ghrelin is a unique substrate for hGOAT within the human proteome, providing further justification for the ghrelin-hGOAT system as a desirable drug target. By defining an array of substrate-enzyme interactions used by hGOAT to bind, recognize, and acylate ghrelin, this study yields novel insight into the character of the hGOAT active site that can serve as a guide toward the rational design of hGOAT inhibitors. [ABSTRACT FROM AUTHOR]

The DEBS1-TE fusion protein is comprised of the loading module, the first two extension modules, and the terminal TE domain of the Saccharopolyspora erythraea 6-deoxyerythronolide B synthase. DEBS1-TE produces triketide lactones that differ on the basis of the starter unit selected by the loading module. Typical fermentations with plasmid-based expression of DEBS1-TE produce a 6:1 ratio of propionate to isobutyrate-derived triketide lactones. Functional dissection of the loading module from the remainder of DEBS1-TE results in 50% lower titers of triketide lactone and a dramatic shift in the production to a 1:4 ratio of propionate to isobutyrate-derived products. A series of radiolabeling studies of the loading module has shown that transfer from the AT to the ACP occurs much faster for propionate than for isobutyrate. However, the equilibrium occupancy of the AT favors isobutyrate such that propionate is outcompeted for ACP occupancy. Thus, propionyl-ACP is the kinetic product, while isobutyryl-ACP is the thermodynamic product. A slowed transfer from the loading domain ACP to first-extension module KS due to functional dissection of DEBS1-TE allows this isobutyryl-ACP-favored equilibrium to be realized and likely accounts for the observed shift in triketide lactone products. [ABSTRACT FROM AUTHOR]