Seeking an ancient enzyme in Methanococcus jannaschii using ORF, a program based on predicted secondary structure comparisons

Abstract

We have developed a simple procedure to identify protein homologs in genomic databases. The program, called ORF, is based on comparisons of predicted secondary structure. Protein structure is far better conserved than amino acid sequence, and structure-based methods have been effective in exploiting this fact to find homologs, even among proteins with scant sequence identity. ORF is a secondary structure-based method that operates solely on predictions from sequence and requires no experimentally determined information about the structure. The approach is illustrated by an example: Thymidylate synthase, a highly conserved enzyme essential to thymidine biosynthesis in both prokaryotes and eukaryotes, is thought to be used by Archaea, but a corresponding gene has yet to be identified. Here, a candidate thymidylate synthase is identified as a previously unassigned open reading frame from the genome of Methanococcus jannaschii, viz., MJ0757. Using primary structure information alone, the optimally aligned sequence identity between MJ0757 and Escherichia coli thymidylate synthase is 7%, well below the threshold of sensitivity for detection by sequence-based methods.

Figure 1

Cartoon of E. coli TS. A ribbon diagram of E. coli TS, with helices in red, strands in yellow, turns in blue, and coil in white (PDB file 2TSC, chain A, 26). The active site cleft, on the left, is shown with bound dUMP (thick wireframe) and anti-folate CB3717 (thin wireframe).

Figure 2

Secondary structure alignment of E. coli TS and MJ0757. Sequence alignment (in single-letter code) generated by orf, based on predicted secondary structure. Observed secondary structure (blue), from x-ray coordinates used in Fig. 1, is shown above the E. coli sequence, with α-helices indicated by hatched boxes and β-strands by arrows. Secondary structure predicted by both gor (25) (blue) and phd (gray) (38) is shown below the MJ0757 sequence. The position of the catalytic cysteine is highlighted in yellow. Residues are numbered for convenience.

Figure 3

Multiple sequence alignment of TS with MJ0757. Optimal alignment of TS sequences B. subtilis, E. coli, S. cerevisiae, L. casei, and MJ0757 was generated with clustal w (27), using default parameters. The catalytic Cys and other residues from Table 1 are highlighted in yellow. The 17 absolutely conserved residues are annotated by an asterisk, and other strongly conserved residues are marked by a dot.

Figure 4

Three-dimensional model of MJ0757. Using the program look 2.0, the MJ0757 sequence was threaded onto the E. coli structure. Side chain placement was refined in look 2.0 and further refined in x-plor (28). The model is visualized in rasmol (53) and color-coded by residue property: hydrophobic (V, I, L, M, F, W, and C), polar (S, T, N, and Q), special backbone (A, P, and G), positively charged (K and R), and negatively charged (D and E). The E. coli x-ray structure (Upper) and MJ0757 modeled structure (Lower) are shown separately for comparison.