Evolution of new function in the GTP cyclohydrolase II proteins of Streptomyces coelicolor

Abstract

The genome sequence of Streptomyces coelicolor contains three open reading frames (sco1441, sco2687, and sco6655) that encode proteins with significant (>40%) amino acid identity to GTP cyclohydrolase II (GCH II), which catalyzes the committed step in the biosynthesis of riboflavin. The physiological significance of the redundancy of these proteins in S. coelicolor is not known. However, the gene contexts of the three proteins are different, suggesting that they may serve alternate biological niches. Each of the three proteins was overexpressed in Escherichia coli and characterized to determine if their functions are biologically overlapping. As purified, each protein contains 1 molar equiv of zinc/mol of protein and utilizes guanosine 5'-triphosphate (GTP) as substrate. Two of these proteins (SCO 1441 and SCO 2687) produce the canonical product of GCH II, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (APy). Remarkably, however, one of the three proteins (SCO 6655) converts GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (FAPy), as shown by UV-visible spectrophotometry, mass spectrometry, and NMR. This activity has been reported for a GTP cyclohydrolase III protein from Methanocaldococcus jannaschii [Graham, D. E., Xu, H., and White, R. H. (2002) Biochemistry 41, 15074-15084], which has no amino acid sequence homology to SCO 6655. Comparison of the sequences of these proteins and mapping onto the structure of the E. coli GCH II protein [Ren, J., Kotaka, M., Lockyer, M., Lamb, H. K., Hawkins, A. R., and Stammers, D. K. (2005) J. Biol. Chem. 280, 36912-36919] allowed identification of a switch residue, Met120, which appears to be responsible for the altered fate of GTP observed with SCO 6655; a Tyr is found in the analogous position of all proteins that have been shown to catalyze the conversion of GTP to APy. The Met120Tyr variant of SCO 6655 acquires the ability to catalyze the conversion of GTP to APy, suggesting a role for Tyr120 in the late phase of the reaction. Our data are consistent with duplication of GCH II in S. coelicolor promoting evolution of a new function. The physiological role(s) of the gene clusters that house GCH II homologues will be discussed.

FIGURE 1

Reactions catalyzed by GCH I, GCH II, and GCH III proteins.

FIGURE 2

Alignment of the E. coli GCH II protein with the proteins that are encoded by three open reading frames that have been annotated as GCH II proteins. The sequence accession numbers are B1277 for the E. coli protein and SCO 1441, SCO 2687, and SCO 6655 for the S. coelicolor proteins. SCO 1441 is a bifunctional protein with an N-terminal DHBP synthase and a C-terminal GCH II domain. For simplicity only the C-terminal region of the protein (starting from amino acid 200) is shown. The boxes denote the cysteine residues that have been shown to be essential for the activity of the E. coli GCH II (20, 21) and coordinate the catalytic zinc divalent metal ion (12). The molecular switch residue that dictates the fate of GTP as shown in SCO 6655 is shown in bold.

FIGURE 3

Spectral changes (A–C) and difference spectra (D) observed with SCO 1441, SCO 2687, and SCO 6655 in the presence of GTP. Each of the assay mixtures contained 0.1 M Tris-HCl (pH 8.0), 5 mM MgCl₂, 0.5 mM DTT, and 0.1 mM GTP in a total volume of 1 mL at 25 °C. The contents of the cuvettes were blanked in the absence of GTP. Spectra were acquired at various times after addition of GTP. The protein concentrations were (A) 7.5 µM SCO 1441, (B) 5.3 µM SCO 2687, and (C) 2.5 µM SCO 6655. The insets show that in each case the rate of the reaction is linear with the concentration of the protein. The difference spectra shown in (D) for SCO 1441 (−), SCO 2687 (•••), and SCO 6655 (---) were obtained by subtracting the spectrum of GTP acquired in the absence of protein from the final spectra that were obtained with each protein. The arrows denote increases and decreases in spectral features.

FIGURE 4

Mass spectral analysis of the products of E. coli GCH II (A), SCO 1441 (B), SCO 2687 (C), and SCO 6655 (D) proteins. The products were isolated as described in Materials and Methods. The mass spectrum of the product of E. coli GCH II is shown for comparison. In some cases, a [M⁻ + Na⁺] complex is also observed.

FIGURE 5

Expanded region of the 2D {¹H-¹³C} HMBC spectrum of FAPy in ²H₂O showing cross-peaks between the formyl proton (H8) and the ¹³C resonance at position 5 of the base. Horizontal lines show the ¹³C shifts of C6 (base) and C1′ (sugar), where cross-peaks would be expected if the formyl group had been located at N9 instead of N7.

FIGURE 6

Active site of E. coli GCH II protein from ref . The protein was cocrystallized with the substrate analogue GMP-CPP. This figure was prepared using PyMol (51) and the PDB structure 2bz0.

FIGURE 7

Difference spectra observed with SCO 6655, SCO 2687, and the Met120Tyr variant of SCO 6655. Each assay mixture contained 0.1 M Tris-HCl (pH 8.0), 5 mM MgCl₂, 0.5 mM DTT, and 85 µM GTP in a total volume of 0.8 mL at 25 °C. Protein concentrations were 1.4, 4.3, and 4.8 µM for SCO 6655, SCO 2687, and the Met120Tyr variant of SCO 6655, respectively. The raw UV-visible spectra that were obtained in this experiment were similar to those shown in Figure 3 (data not shown). Spectral differences were calculated from the initial and final spectra obtained immediately and 30 min after addition of substrate.

FIGURE 8

Proposed catalytic mechanism for GCH II. For simplicity, the hydrolysis of the pyrophosphate moiety, which occurs at a distant location, is not shown. The hydrolysis of C8 of GTP to generate APy has been proposed to occur in two half-reactions. Our data support the notion that the Tyr residue (not shown) in the active site of the protein (see Figure 6) acts in the second half-reaction.