HMP binding protein ThiY and HMP-P synthase THI5 are structural homologues

Abstract

The ATP-binding cassette transporter system ThiXYZ transports N-formyl-4-amino-5-(aminomethyl)-2-methylpyrimidine (FAMP), a thiamin salvage pathway intermediate, into cells. FAMP is then converted to 4-amino-5-(hydroxymethyl)-2-methylpyrimidine (HMP) and recycled into the thiamin biosynthetic pathway. ThiY is the periplasmic substrate binding protein of the ThiXYZ system and delivers the substrate FAMP to the transmembrane domain. We report the crystal structure of Bacillus halodurans ThiY with FAMP bound at 2.4 Å resolution determined by single-wavelength anomalous diffraction phasing. The crystal structure reveals that ThiY belongs to the group II periplasmic binding protein family. The closest structural homologues of ThiY are periplasmic binding proteins involved in alkanesulfonate/nitrate and bicarbonate transport. ThiY is also structurally homologous to thiamin binding protein (TbpA) and to thiaminase-I. THI5 is responsible for the synthesis of 4-amino-5-(hydroxymethyl)-2-methylpyrimidine phosphate in the thiamin biosynthetic pathway of eukaryotes and is approximately 25% identical in sequence with ThiY. A homology model of Saccharomyces cerevisiae THI5 was generated on the basis of the structure of ThiY. Many features of the thiamin pyrimidine binding site are shared between ThiY and THI5, suggesting a common ancestor.

Figure 1

Monomeric structure of ThiY. (A) The cartoon diagram of ThiY with the secondary structure elements labeled. The 3₁₀ helices are labeled as η. FAMP is shown as sticks. Carbon atoms in FAMP are colored green, nitrogen atoms are colored blue and oxygen atoms are colored red. (B) The topology diagram of ThiY showing the domain organization.

Figure 2

Dimeric form of ThiY looking down the two-fold axis. The monomers are colored blue and gray. FAMP bound in the active site is shown as sticks.

Figure 3

FAMP binding site in ThiY. (A) Stereoview of the FAMP binding site. FAMP has carbon atoms colored green. Water molecules are shown as red spheres and hydrogen bonds are shown as dashed lines. (B) Schematic representation of the binding site showing the key hydrogen bonding and stacking interactions.

Figure 4

Structural homologues for ThiY. Comparisons of ThiY with (A) SsuA, (B) thiaminase-I and (C) TbpA. ThiY carbon atoms are colored green and carbon atoms of the compared structure are colored cyan. The ligands are shown in ball and stick. (D) Superposition of the binding sites of ThiY and TbpA. Hydrophobic residues involved in stacking of TMP in TbpA are shown.

Figure 5

Homology model of yeast THI5. Stereo ribbon diagram showing the Cα trace of both the domains. The [4Fe-4S] cluster, Trp12, and FAMP (superimposed from ThiY) are shown as sticks. The CCCXC motif binding the cluster is located in the cleft on the other side of the active site.

Scheme 1

Scheme 2