Serine binding

Abstract: Serine acetyltransferase (SAT, EC 2.3.1.30) catalyzes the CoA-dependent acetylation of the side chain hydroxyl group of l-serine to form O-acetylserine, as the first step of a two-step biosynthetic pathway in bacteria and plants leading to the formation of l-cysteine. This reaction represents a key metabolic point of regulation for the cysteine biosynthetic pathway due to its feedback inhibition by cysteine. We have determined the X-ray crystal structure of Haemophilus influenzae SAT in complexes with CoA and its cysteine feedback inhibitor. The enzyme is a 175 kDa hexamer displaying the characteristic left-handed parallel beta-helix (LbetaH) structural domain of the hexapeptide acyltransferase superfamily of enzymes. Cysteine is bound in a crevice between adjacent LbetaH domains and underneath a loop excluded from the coiled LbetaH. The proximity of its thiol group to the thiol group of CoA derived from superimposed models of the cysteine and CoA complexes confirms that cysteine is bound at the active site. Analysis of the contacts of SAT with cysteine and CoA and the conformational differences that distinguish these complexes provides a structural basis for cysteine feedback inhibition, which invokes competition between cysteine and serine binding and a cysteine-induced conformational change of the C-terminal segment of the enzyme that excludes binding of the cofactor.

Abstract: Ligand binding to the serine receptor of Escherichia coli has been studied using isothermal titration calorimetry. Bacterial inner membranes enriched in the serine receptor (Tsr) were titrated as sonicated membrane samples and after solubilization in octyl beta-D-glucopyranoside (OG) to determine the number of moles of ligand bound per mole of receptor (n), the binding constant (Ka), and the enthalpy of binding (delta H) of serine to the receptor. The n value for serine binding to OG-solubilized Tsr protein (n = 0.5) was consistent with one molecule of serine binding to a receptor dimer, but in sonicated inner membrane samples, the n value was smaller (n approximately equal to 0.25), indicating that not all of the binding sites were accessible to added serine. At 7 and 27 degrees C, the values for Ka and delta H were equivalent for the membrane and OG-solubilized samples and were found to be 4.7 x 10(4) M-1 and -15 kcal/mol, and 3.6 x 10(4) M-1 and -18 kcal/mol, respectively. The influence of covalent modification at the sites of methylation on the affinity of the receptor for serine was also investigated, and found to have only a modest effect. The property of half-site saturation is suggestive of models for transmembrane signaling where the receptor subunit interactions are modulated by ligand binding.

Abstract: We used freeze trapping to stabilize the Michaelis complex of wild-type tryptophan synthase and the α-subunit substrate indole-3-glycerol phosphate (IGP) and determined its structure to 1.8 A resolution. In addition, we determined the 1.4 A resolution structure of the complex with indole-3-propanole phosphate (IPP), a noncleavable IGP analogue. The interaction of the 3‘-hydroxyl of IGP with the catalytic αGlu49 leads to a twisting of the propane chain and to a repositioning of the indole ring compared to IPP. Concomitantly, the catalytic αAsp60 rotates resulting in a translocation of the COMM domain [βGly102-βGly189, for definition see Schneider et al. (1998) Biochemistry 37, 5394−5406] in a direction opposite to the one in the IPP complex. This results in loss of the allosteric sodium ion bound at the β-subunit and an opening of the β-active site, thereby making the cofactor pyridoxal 5‘-phosphate (PLP) accessible to solvent and thus serine binding. These findings form the structural basis for the inform...