Biochemical and thermodynamic analyses of Salmonella enterica Pat, a multidomain, multimeric N(ε)-lysine acetyltransferase involved in carbon and energy metabolism

Abstract

In the bacterium Salmonella enterica, the CobB sirtuin protein deacetylase and the Gcn5-related N(ε)-acetyltransferase (GNAT) Pat control carbon utilization and metabolic flux via N(ε)-lysine acetylation/deacetylation of metabolic enzymes. To date, the S. enterica Pat (SePat) acetyltransferase has not been biochemically characterized. Here we report the kinetic and thermodynamic characterization of the SePat enzyme using two of its substrates, acetyl coenzyme A (Ac-CoA) synthetase (Acs; AMP forming, EC 6.2.1.1) and Ac-CoA. The data showed typical Michaelis-Menten kinetic behavior when Ac-CoA was held at a saturating concentration while Acs was varied, and a sigmoidal kinetic behavior was observed when Acs was saturating and the Ac-CoA concentration was varied. The observation of sigmoidal kinetics and positive cooperativity for Ac-CoA is an unusual feature of GNATs. Results of isothermal titration calorimetry (ITC) experiments showed that binding of Ac-CoA to wild-type SePat produced a biphasic curve having thermodynamic properties consistent with two distinct sites. Biphasicity was not observed in ITC experiments that analyzed the binding of Ac-CoA to a C-terminal construct of SePat encompassing the predicted core acetyltransferase domain. Subsequent analytical gel filtration chromatography studies showed that in the presence of Ac-CoA, SePat oligomerized to a tetrameric form, whereas in the absence of Ac-CoA, SePat behaved as a monomer. The positive modulation of SePat activity by Ac-CoA, a product of the Acs enzyme that also serves as a substrate for SePat-dependent acetylation, is likely a layer of metabolic control. IMPORTANCE For decades, N(ε)-lysine acetylation has been a well-studied mode of regulation of diverse proteins involved in almost all aspects of eukaryotic physiology. Until recently, N(ε)-lysine acetylation was not considered a widespread phenomenon in bacteria. Recent studies have indicated that N(ε)-lysine acetylation and its impact on cellular metabolism may be just as diverse in bacteria as they are in eukaryotes. The S. enterica Pat enzyme, specifically, has recently been implicated in the modulation of many metabolic enzymes. Understanding the molecular mechanisms of how this enzyme controls the activity of diverse enzymes by N(ε)-lysine acetylation will advance our understanding of how the prokaryotic cell responds to its changing environment in order to meet its metabolic needs.

FIG 1

SePat is a multidomain protein that belongs to the GNAT superfamily of enzymes. SePat is predicted to be a multidomain protein (Conserved Domain Database [51] search) that has a C-terminal Ac-CoA binding fold whose predicted structure belongs to the large GNAT superfamily of acetyltransferases. N terminal to this domain is another predicted domain having high similarity to the acyl CoA synthetase (NDP-forming) superfamily of enzymes.

FIG 2

Initial velocity of SePat in response to Acs_C and Ac-CoA substrate concentrations. (A) The graph on the left shows the substrate saturation curve of the SePat-dependent acetylation reaction velocity in response to various Acs substrate concentrations. The curve is hyperbolic with an r² value of 0.98 and was determined from three independent experiments. The graph on the right is a double-reciprocal plot of the kinetic data. SePat was present at 30 nM, and Ac-CoA (100 µM) was used at a saturating concentration. (B) The graph on the left shows the substrate saturation curve of the SePat-dependent acetylation reaction velocity in response to various Ac-CoA substrate concentrations. The curve was best fitted to a sigmoidal curve with an r² value of 0.98 and was determined from three independent experiments. The graph on the right is a double-reciprocal plot of the kinetic data indicating a concave curve. SePat enzyme was present at 15 nM, and Acs_C was present at a saturating concentration (400 µM).

FIG 3

ITC profile of Ac-CoA binding to SePat. The binding isotherm for Ac-CoA is biphasic. (Top) Raw data from titration of consecutive 5-µl injections of Ac-CoA (750 µM) into full-length SePat (50 µM), represented as the heat change (µcal/s) upon injection over time. (Bottom) Binding isotherm obtained by integration of the raw data (reported as kcal/mol of Ac-CoA injected). The solid line represents the best-fit curve generated from a two-site binding model.

FIG 4

ITC profile of Ac-CoA binding to SePat_AT. (Top) Raw data from titration of consecutive 5-µl injections of Ac-CoA (750 µM) into SePat_AT (50 µM), represented as the heat change (µcal/s) upon injection over time. (Bottom) Binding isotherm obtained by integration of the raw data (reported as kcal/mol of Ac-CoA injected). The solid line represents the best-fit curve generated from a one-site binding model.

FIG 5

Oligomeric state of Pat in the presence and absence of Ac-CoA. The molecular mass of Pat in solution was estimated by gel filtration. At a flow rate of 0.3 ml/min, Pat (2.5 µM) in the absence of Ac-CoA eluted at 40 ± 0.1 min (red chromatogram) and had an apparent molecular mass of 86 ± 2 kDa compared to the elution times of known molecular masses. In the presence of 50 µM Ac-CoA, Pat eluted at 30 ± 0.1 min (blue chromatogram) and had an apparent molecular mass of 428 ± 5 kDa.