The entire flap domain region superimposed on the final electron density 2F_o − F_c map, contoured at the 1.0 σ level. This figure was prepared with the program setor (20).

Ball and stick diagrams of the HMG-CoA (a and b) and mevalonate (c) substrates in the active site of HMG-CoA reductase. The substrates are shown as stick models with C = orange, N = blue, O = red, and S = yellow. (a) Critical contacts with the enzyme (silver bonds) at the site of cleavage of HMG-CoA (green bonds). The thioester oxygen of the substrate points directly at Lys267, making a strong hydrogen bond with this newly identified catalytic residue. The contact between the catalytic His381 and His385, proposed to aid in the protonation of the CoAS⁻ group, also is shown. (b) The hydrogen bonding network that holds Lys267 in place. Shown are the first shell of hydrogen bond contacts that involve primarily positive and negatively charged residues. (c) Critical contacts of the catalytic residues with the mevalonate substrate. In this ternary complex, the tetrahedral carbon (C-5) points its OH toward one of the Glu83 carboxylic oxygens, suggesting a change in the primary contact of that oxygen after isomerization. This also suggests that Glu83 may be involved in accepting a proton from mevaldyl-CoA, which should have the same tetrahedral configuration for C-5. This figure was prepared with setor (20).

(A) Ribbon diagram of the HMG-CoA reductase dimer with HMG-CoA and NAD⁺ bound. The view looks directly into the active site cavity between the two monomers shown in red and gray. The flap domain, in yellow, extends from residue 375 in the red monomer in three helical segments (marked 1–3) to the C-terminal residue of the molecule, residue 428. The substrates are shown as stick models with C = green, N = blue, O = red, P = gray, and S = yellow. Both substrates bind in an extended conformation with, HMG-CoA binding to the large domain of the red monomer on the left and NAD⁺ binding to the small domain of the gray monomer on the right. (B and C) Close-up views of the active site in the ternary complexes of HMG-CoA reductase with HMG-CoA/NAD⁺ and mevalonate/ NADH, respectively. The catalytic His381 side chain in the closed flap is shown as a ball and stick model, positioned near the sulfur of the HMG-CoA in B. The similar position of the HMG moiety of HMG-CoA and of mevalonate in these complexes is readily seen. The NAD(H) nicotinamide ring in the anti/pro-S configuration lays side-by-side with the substrate, positioned for hydride transfer. This figure was prepared with the program ribbons (19).

Scheme of the reaction mechanism proposed for P. mevalonii HMG-CoA reductase. The three steps of the reaction from HMG-CoA to mevalonate are shown, and the roles proposed for the key catalytic residues Lys267, Asp283, Glu83, and His381 are indicated.

Alignment of HMG-CoA reductase sequences around the critical catalytic residues. Four regions of sequence around the catalytic residues of P. mevalonii HMG-CoA reductase (Glu83, Lys267, Asp283, and His381) are shown in comparison to selected sequences from a variety of biosynthetic HMG-CoA reductases. The numbering for P. mevalonii is shown in bold at the bottom. The numbering at the top of the figure is that for the human enzyme, given as a reference. Catalytic residues are indicated by white on black lettering. Conserved residues are shaded in gray. Site-directed mutagenesis of the P. mevalonii and Syrian hamster enzymes have confirmed that the aligned residues are catalytic homologues.