Structural genomics of enzymes involved in sterol/isoprenoid biosynthesis

Abstract

X-ray structures of two enzymes in the sterol/isoprenoid biosynthesis pathway have been determined in a structural genomics pilot study. Mevalonate-5-diphosphate decarboxylase (MDD) is a single-domain alpha/beta protein that catalyzes the last of three sequential ATP-dependent reactions which convert mevalonate to isopentenyl diphosphate. Isopentenyl disphosphate isomerase (IDI) is an alpha/beta metalloenzyme that catalyzes interconversion of isopentenyl diphosphate and dimethylallyl diphosphate, which condense in the next step toward synthesis of sterols and a host of natural products. Homology modeling of related proteins and comparisons of the MDD and IDI structures with two other experimentally determined structures have shown that MDD is a member of the GHMP superfamily of small-molecule kinases and IDI is similar to the nudix hydrolases, which act on nucleotide diphosphatecontaining substrates. Structural models were produced for 379 proteins, encompassing a substantial fraction of both protein superfamilies. All three enzymes responsible for synthesis of isopentenyl diphosphate from mevalonate (mevalonate kinase, phosphomevalonate kinase, and MDD) share the same fold, catalyze phosphorylation of chemically similar substrates (MDD decarboxylation involves phosphorylation of mevalonate diphosphate), and seem to have evolved from a common ancestor. These structures and the structural models derived from them provide a framework for interpreting biochemical function and evolutionary relationships.

Figure 1

Pathways for biosynthesis of isopentenyl diphosphate. Isopentenyl diphosphate, the central intermediate in sterol/isoprenoid biosynthesis, is produced by two independent pathways, which have different evolutionary distributions (39, 40).

Figure 2

MDD sequence alignment. Secondary structural elements of S. cerevisiae MDD are shown with cylinders (α-helices) and arrows (β-strands). Gray dots denote poorly resolved residues in the final electron density map. Color-coding denotes sequence conservation among MDDs (white → green ramp, 30 → 100% similarly). Red box denotes the putative ATP-binding P loop.

Figure 3

IDI sequence alignment. Secondary structural elements, poorly resolved residues, and sequence conservation are denoted as in Fig. 2. #, metal-binding residues; *, conserved residues in the cleft; o = the active-site Cys. Some of the N- and C-terminal residues of IDI sequences other than E. coli have been excluded for clarity.

Figure 4

S. cerevisiae MDD and M. jannaschii HSK. Ribbon drawings of MDD (A) and HSK (B) in the same orientation. The two-fold rotational symmetry axis that generates the MDD homodimer is indicated. Putative P loops are colored red.

Figure 5

E. coli IDI and MutT. Ribbon drawings IDI (A) and MutT (B) in the same orientation. Ball-and-stick representations of the divalent metal ion and putative active-site residues are given for IDI (atom type code: C, green; S, yellow; N, blue; O, red; Mn²⁺, gray). The positions of the equivalent conserved motifs of the two proteins are colored magenta.