Proposed catalytic mechanism of SPT. a, the internal aldimine (holo-SPT) is displaced by l-serine to form the external adlimine; b, binding of second substrate palmitoyl-CoA causes conformational change to give the Dunathan intermediate; c, formation of the quinonoid by deprotonation of Cα hydrogen; d, thioester bond hydrolysis and release of CoASH to give β-keto acid intermediate; e, decarboxylation to form KDS product quinonoid; f, reprotonation to form KDS product external aldimine; g, KDS product release and reformation of the holo-form. The l-serine external aldimine has been trapped in the current study. The dotted line shows that steps e and f may be bypassed by an alternative mechanism that does not proceed via a product quinonoid (see Ref. 21).

Assay of SPT activity. A and B, TLC of radiolabeled products obtained by SPT-catalyzed conversion of l-serine and palmitoyl-CoA to [¹⁴C]KDS. Recombinant holo-SPT was incubated with U-¹⁴C-labeled l-serine and palmitoyl-CoA for 20 min at 37 °C. The assay was stopped by the addition of NH₄OH and extracted with an equal volume of chloroform/methanol (2:1, v/v). Organic phases were removed, dried, separated by TLC, and analyzed by a PhosphorImager. The arrow marks the position of the standard KDS. A, products observed after reaction of the SPT N100C (lane 1), SPT N100W (lane 2), SPT N100Y (lane 3), and SPT WT (lane 4). B, products observed after reaction of the SPT WT (lane 1), SPT R378A (lane 2), and SPT R378N (lane 3). C, kinetic analysis of wild-type SPT using a spectrophotometric assay. i, initial rates of CoASH release measured spectrophotometrically at 412 nm and at several final concentrations of l-serine: 6.25 mm (a), 1.56 mm (b), 0.78 mm (c), 0.39 mm (d), and 0.12 mm (e). Each assay contained 0.16 μm enzyme, 250 μm palmitoyl-CoA, and 0.2 mm DTNB. ii, the initial rates were converted to k_cat^app values and plotted against l-serine concentration to determine the K_m and k_cat parameters. iii, complete condensation of 5 μm palmitoyl-CoA by 0.57 μm wild-type SPT in the presence of 100 mm l-serine. k_cat/K_m for palmitoyl-CoA was calculated by dividing k_obs by the enzyme concentration.

Structure of the SPT·l-serine external aldimine complex. A, overall structure of the SPT dimer showing one monomer in light blue and the other in purple. The residues Lys²⁶⁵ and Asn¹⁰⁰ from the light blue monomer are shown in stick form and are colored yellow. The equivalent residues from the purple monomer are colored green. This clearly shows that the Asn¹⁰⁰ from one monomer interacts with the active site of the other monomer. The PLP·l-ser external aldmine is also shown in stick form. B, a stereo image of an F_o − F_c electron density map contoured at 3 σ around the PLP·l-ser external aldimine found in the native SPT. The phases were calculated from a model that had never included the PLP or aldimine.

Schematic representation of the residues involved in SPT catalysis, conformations of the external aldimines, and roles of active site arginine residues in controlling. A, key residues involved in forming the active site are highlighted. The Arg³⁷⁸ side chain is in the swung out conformation and interacts with Gln³⁵⁷. B, binding of l-Ser leads to PLP·l-Ser external aldimine formation. The residues observed in the wild-type crystal structure are shown. The carboxylate of l-Ser interacts with the side chains of His¹⁵⁹ and Arg³⁷⁸, which is now in the swung in conformation. Rotation around the Cα–Nα bond (arrow) brings the intermediate into the desired Dunathan conformation shown in C. The carboxylate is shown to possibly interact with Arg³⁹⁰, and the Cα-H is now perpendicular to the PLP ring. This is then deprotonated by Lys²⁶⁵ to form the quinonoid. D, stereo image of a model of the external aldimine Dunathan conformation using the external aldimine structure.

UV-visible analysis of SPT wild-type and mutants. Absorbance spectra of S. paucimobilis SPT wild-type (A) and mutants N100C (B), N100W (C), and N100Y (D). In spectrum A the enolimine (335 nm) and ketoenamine (420 nm) forms of the external aldimine are drawn. The solid line in each spectrum is the holo-form of the enzyme (10 μm SPT, 20 mm potassium phosphate buffer (pH 7.5), 150 mm NaCl, 25 °C). Increasing concentrations of l-serine were added (0, 0.1, 0.5, 1, 2, 5, 10, 20, and 40 mm; dotted and dashed lines), and the spectrum was recorded after 15 min. For the SPT N100C, N100W, and N100Y spectra the final concentrations of l-serine were 0, 0.1, 0.5, 1, 2, 5, 10, 20, 40, 60, and 80 mm. AU, absorbance units.

Quinonoid formation in the wild-type SPT, N100Y, and R378A mutants. UV-visible spectra of the SPT wild type (A), N100Y (B), and R378A (C) external aldimine forms after titration of the thioether analog (S-(2-oxoheptadecyl)-CoA) of palmitoyl-CoA. Incubations were carried out with 5 μm enzyme, 45 mm l-serine, 1.4 mm analog, 20 mm potassium phosphate buffer (pH 7.5), 150 mm NaCl for 15 min at 25 °C. The solid line in each spectrum represents the holo-form; the dotted line is the external aldimine form after the addition of l-ser; and the dashed line is after the addition of the analogue. An absorbance maximum at 495 nm (marked with an arrow), attributed to a quinonoid species (50), is clearly observed in the wild-type enzyme, but only a broad shoulder is produced in the N100Y mutant, and no peak is observed for the R378A mutant. AU, absorbance units.

Structure of the SPT·l-serine external aldimine complex and impact of the N100Y HSAN1 mutation. A, a stereo image showing residues involved in binding the PLP·l-ser external aldimine complex are shown. His¹⁵⁹, Asp²³¹, His²³⁴, Thr²⁶², Lys²⁶⁵, and Arg³⁷⁸ are from monomer A (light blue) in yellow stick format. Residues Thr²⁹⁴ and Asn¹⁰⁰ are from monomer B (purple), are colored green, and are annotated with an asterisk. Residues His¹⁵⁹ and Arg³⁷⁸ interact with the carboxylate group of the l-serine; Lys²⁶⁵ (denoted by dashed lines) interacts with the hydroxyl group of the l-serine; and His²³⁴ interacts with the hydroxyl group of the PLP. Residue Asp²³¹ binds to the nitrogen atom of the PLP cofactor, and the side chain of Asn¹⁰⁰ (from monomer B) interacts with the backbone of Lys²⁶⁵ of the opposite monomer A. B, a stereo image of an overlay of the external aldimine forms of the wild-type and N100Y. The wild-type structure has been rendered with monomer A colored with a blue backbone and yellow side chains, and monomer B is shown with a purple backbone and green side chains. In the N100Y mutant, the monomer A backbone and side chains are colored gold, and monomer B is colored with a red backbone and salmon pink side chains. The N100 side chain from monomer B of the wild-type SPT (green stick, asterisk) interacts with residues from monomer A. However, in the SPT N100Y mutant, the Tyr¹⁰⁰ side chain has flipped into a pocket formed by displacement of Phe¹⁰⁹ of the same monomer B, residue Asn¹⁰⁶ from monomer B has also undergone a large shift, and residue Asn³⁷⁵ from monomer A has also changed. The backbone containing residue Tyr¹⁰⁰ has also undergone a large conformational change. C, a stereo image of an overlay of the external aldimine forms of the wild-type and N100Y showing the impact of mutation on Arg³⁷⁸ and the PPATP loop. The coloring is the same as in B. In wild-type SPT, residue Arg³⁷⁸ (side chain yellow on the blue backbone) is in the swung in position and interacts with the l-Ser carboxylic acid. In the SPT N100Y mutant, the “PPATP loop” and the Arg³⁷⁸ residue (backbone and side chain colored gold) are in the “swung out” conformation observed in the wild-type internal aldimine form. The interaction between Arg³⁷⁸ and Gln³⁵⁷ (both in gold) and the different positions of the Asn¹⁰⁰ and Tyr¹⁰⁰ residues are shown.