PMC

Substrates utilized by MIBS and their unreactive analogues.

Stereographic cut-away view of superimposed active site contours of MIBS (green) and BPPS (pink, PDB ID: 1N20). Geranyl diphosphate analogues GSPP and AGPP are shown as ball-and-stick figures in darker shades, and selected amino acid side chains are indicated.

The 35-kDa C-terminal catalytic domain of MIBS adopts the α-helical class I terpenoid synthase fold as shown, and the 13-kDa proline-rich N-terminal domain is disordered and hence not shown. The N- and C-termini are labeled with black letters, and the D¹⁹⁷DCYCED and N³⁴⁵DLYSYTKE metal-binding motifs are red and orange, respectively. Disordered polypeptide segments are indicated by dotted lines. The active site is indicated by the binding of 2FGPP (stick figure) and two Mg²⁺ ions (small magenta spheres).

Dimeric quaternary structures of MIBS, farnesyl diphosphate synthase from Gallus gallus (FPPS; PDB ID: 1UBW), and (+)-bornyl diphosphate synthase from Salvia officinalis (BPPS; PDB ID: 1N20). The catalytic C-terminal domain, which forms the dimerization interface, is dark/light blue; the N-terminal domain of BPPS is dark/light green. A red arrow indicates the mouth of each active site. The active sites in the MIBS dimer are oriented in parallel fashion, like those in the FPPS dimer. The active sites in the BPPS dimer are oriented in antiparallel fashion, which is the case for all structures of terpenoid cyclase dimers determined to date.

Proposed cyclization mechanisms of MIBS (), (+)-bornyl diphosphate synthase (-), and endo-fenchol synthase (). Common to each mechanism are conformational changes (red arrows) in the (3R)-linalyl diphosphate intermediate (or its 2-methyl derivative) leading to the cisoid-anti, endo-boat conformation required for cyclization. The conversion of the (4R)-α-terpinyl cation to (+)-bornyl diphosphate could occur in a single, concerted step or through a nonclassical carbonium ion to avoid formation of the 2-bornyl cation, a relatively unstable secondary carbocation (, , ).

Comparison of MIBS and FPPS dimer assembly. (a) Superposition of the FPPS dimer (orange/tan) on one monomer of MIBS (blue); for reference, active sites are indicated by bound ligands. Helices A and B are elongated in FPPS and guide dimer assembly. The protrusion of helix D2 and the D1-D2 loop would block the comparable assembly of the MIBS dimer. (b) Superposition of the MIBS dimer (blue/light blue) on one monomer of FPPS (orange); for reference, active sites are indicated by bound ligands. The protruding helix D2 and the D1-D2 loop help define a surface for dimer assembly that is absent in FPPS. (c) Schematic diagram of the class I terpenoid synthase fold showing the general interfaces utilized by various members of the class for dimerization. Major helices are indicated by circles colored in “rainbow” sequence from the N-terminus (blue) to the C-terminus (red). Circles with solid colors represent helices with their C-termini directed toward the viewer; those with cross-hatching represent helices that are directed away from the viewer. Metal-binding motifs on helices D and H are also indicated. If a second domain is present, such as the proline-rich domain of MIBS or the non-functional N-terminal domain of BPPS, it would interact with the side opposite that of the dimerization surface, as indicated.

Substrate analogue binding to MIBS. (a) Simulated annealing omit maps showing the binding of 2FGPP (contoured at 4σ) and two Mg²⁺ ions (contoured at 4σ) in the active site of MIBS. Weak electron density for Mg²⁺_C suggests partial occupancy. Side chains of key residues in the D¹⁹⁷DCYCED and N³⁴⁵DLYSYTKE motifs are displayed as stick figures and metal coordination interactions are indicated by green dotted lines. Disordered polypeptide segments appear as broken lines. (b) Simulated annealing omit maps showing the binding of GSPP (contoured at 4σ) and two Mg²⁺ ions (contoured at 4σ) in the active site of MIBS. Side chains of key residues in the D¹⁹⁷DCYCED and N³⁴⁵DLYSYTKE motifs are displayed as stick figures and metal coordination interactions are indicated by green dotted lines. Disordered polypeptide segments appear as broken lines. (c) Stereoview of the MIBS•2FGPP complex; for clarity, only one carbon atom of the isoprenoid group is shown to indicate its connectivity with the diphosphate group. Selected active site residues are indicated; metal coordination and hydrogen bond interactions are indicated by green lines and red dashed lines, respectively. Atoms are color-coded as follows: carbon = dark gray, nitrogen = blue, oxygen = red, sulfur = yellow, phosphorus = orange; Mg²⁺ ions and water molecules appear as magenta spheres and red spheres, respectively. (d) Stereoview of the BPPS•AGPP complex (gray, PDB 1N20); for clarity, only one carbon atom of the isoprenoid group is shown to indicate its connectivity with the diphosphate group. Atomic color coding is the same as in (c) except that carbon = light gray. In contrast with the MIBS•2FGPP complex, the BPPS•AGPP complex contains a complete trinuclear metal cluster.