Crystal structure of glutamate-1-semialdehyde aminomutase: an alpha2-dimeric vitamin B6-dependent enzyme with asymmetry in structure and active site reactivity

Abstract

The three-dimensional structure of glutamate-1-semialdehyde aminomutase (EC 5.4.3.8), an alpha2-dimeric enzyme from Synechococcus, has been determined by x-ray crystallography using heavy atom derivative phasing. The structure, refined at 2.4-A resolution to an R-factor of 18.7% and good stereochemistry, explains many of the enzyme's unusual specificity and functional properties. The overall fold is that of aspartate aminotransferase and related B6 enzymes, but it also has specific features. The structure of the complex with gabaculine, a substrate analogue, shows unexpectedly that the substrate binding site involves residues from the N-terminal domain of the molecule, notably Arg-32. Glu-406 is suitably positioned to repel alpha-carboxylic acids, thereby suggesting a basis for the enzyme's reaction specificity. The subunits show asymmetry in cofactor binding and in the mobilities of the residues 153-181. In the unliganded enzyme, one subunit has the cofactor bound as an aldimine of pyridoxal phosphate with Lys-273 and, in this subunit, residues 153-181 are disordered. In the other subunit in which the cofactor is not covalently bound, residues 153-181 are well defined. Consistent with the crystallographically demonstrated asymmetry, a form of the enzyme in which both subunits have pyridoxal phosphate bound to Lys-273 through a Schiff base showed biphasic reduction by borohydride in solution. Analysis of absorption spectra during reduction provided evidence of communication between the subunits. The crystal structure of the reduced form of the enzyme shows that, despite identical cofactor binding in each monomer, the structural asymmetry at residues 153-181 remains.

Figure 1

Intermediates in the conversion of GSA to 5-aminolevulinate. R, (CH₂)₂COO⁻; Py, the 5′-phosphopyridoxyl group of the cofactor.

Figure 2

Schematic drawing of the secondary structure of GSA-AT. Rectangles and arrows represent α-helices and β-strands, respectively. Secondary structure definitions were made with the program dssp (18).

Figure 3

Stereoview of GSA-AT showing the overall fold and the secondary structure. The cofactor is shown in ball and stick. The view is down the 2-fold symmetry axis of the dimer. Subunit A is in yellow and subunit B is in green. Residues 153–181 are shown as blue in subunit A and as red in subunit B, but it should be noted that in subunit B, this part of the chain is disordered and does not have the structure indicated.

Figure 4

Stereoviews of superpositions of the active sites of the two subunits. The bonds of subunits A and B are shown in yellow and cyan, respectively, except for residues 303–305 which are shown in green (subunit A) and blue (subunit B). (A) Wild-type enzyme. (B) The enzyme after reduction of the Schiff base double bond by cyanoborohydride.

Figure 5

Changes in absorption spectrum during reduction. The enzyme (37 μM) was reacted with NaBH₃CN (0.1 M) in 0.1 M tricine (pH 7.8, 37°C). Absorption spectra were taken at 5-s intervals. (Upper) Absorbance changes observed at single wavelengths of 418 nm (•) and 370 nm (▴). (Lower) Spectra taken (a) at 5-s intervals from the beginning of the reaction and (b) at 50-s intervals 250 s after the start of reaction.

Figure 6

Active site of subunit A with gabaculine noncovalently bound, superimposed on a 3.0-Å resolution omit map, contoured at 4 σ.