Defining critical residues for substrate binding to 1-deoxy-D-xylulose 5-phosphate synthase--active site substitutions stabilize the predecarboxylation intermediate C2α-lactylthiamin diphosphate

Abstract

1-Deoxy-D-xylulose 5-phosphate (DXP) synthase catalyzes the formation of DXP from pyruvate and D-glyceraldehyde 3-phosphate (GraP) in a thiamin diphosphate-dependent manner, and is the first step in the essential pathway to isoprenoids in human pathogens. Understanding the mechanism of this unique enzyme is critical for developing new anti-infective agents that selectively target isoprenoid biosynthesis. The present study used mutagenesis and a combination of protein fluorescence, CD and kinetics experiments to investigate the roles of Arg420, Arg478 and Tyr392 in substrate binding and catalysis. The results support a random sequential, preferred order mechanism, and predict that Arg420 and Arg478 are involved in binding of the acceptor substrate, GraP. D-Glyceraldehyde, an alternative acceptor substrate lacking the phosphoryl group predicted to interact with Arg420 and Arg478, also accelerates decarboxylation of the predecarboxylation intermediate C2α-lactylthiamin diphosphate (LThDP) on DXP synthase, indicating that this binding interaction is not absolutely required, and that the hydroxyaldehyde sufficiently triggers decarboxylation. Unexpectedly, Tyr392 contributes to GraP affinity, and is not required for LThDP formation or its GraP-promoted decarboxylation. Time-resolved CD spectroscopy and NMR experiments indicate that LThDP is significantly stabilized on R420A and Y392F variants as compared with wild-type DXP synthase in the absence of acceptor substrate, but these substitutions do not appear to affect the rate of GraP-promoted LThDP decarboxylation in the presence of high levels of GraP, and LThDP formation remains the rate-limiting step. These results suggest a role of these residues in promoting GraP binding, which in turn facilitates decarboxylation, and also highlight interesting differences between DXP synthase and other thiamin diphosphate-dependent enzymes.

Keywords: CD; DXP synthase; enzymology; protein fluorescence; thiamin.

Figure 1

The MEP pathway for IDP and DMADP biosynthesis.

Figure 2

Active site residues of interest highlighted on D. radiodurans DXP synthase [6]. R480, R423 and Y395 correspond to R478, R420 and Y392, respectively, on E. coli DXP synthase.

Figure 3

Km determinations for R478A (a-b), R420A (c), and Y392F (d-e) DXP synthase variants using the IspC coupled assay.

Figure 4

Determination of Km D-GAP on R478A, R420A and Y392F using CD.

Figure 5

Substrate binding analysis of DXP synthase by intrinsic protein fluorescence. Minimal change in fluorescence is observed upon titration of DXP synthase with water (a and b). Titration of DXP synthase with pyruvate (c and d) or D-GAP (e and f) results in measurable fluorescence quenching. D-GAP titration of R478A (g and h) and R420A (i and j) results in negligible changes in fluorescence.

Figure 6

a) Formation of 1′, 4′–iminopyrimidyl-LThDP (315 nm) from pyruvate (10-500 μM) by R478A DXP synthase (29.6 0M) at 5 °C; b) Pre-steady state analysis of LThDP formation and decarboxylation under single turnover conditions on R478A (30 0M) with pyruvate (15 0M), in the absence of D-GAP at 6 °C. Data were fit to equation 2 (see text); c) Pre-steady state analysis of LThDP decarboxylation (k2) and resynthesis (k1) on R478A (24 0M was pre-mixed with 150 μM pyruvate to form LThDP) in the presence of 1 mM D-GAP at 6 °C (inset) fitting the expansion of early behavior to equation 4.

Figure 7

NMR detection of [1-13C]-DXP formation by R478A DXP synthase during pre-steady state, D-GAP promoted decarboxylation of pre-formed [C2β-13C]-LThDP. a) Only LThDP is present on R478A DXP synthase 5 seconds after addition of D-GAP, as determined by examination of the C6′-H region in the 1H NMR spectrum; b) Detection of 13CH3 labeled groups: [1-13C]-DXP (28.5 %), [C2β-13C]-LThDP (35.5 %) and [3-13C]-pyruvate (23.3 %) by 1D gCHSQC spectroscopy (decoupled) 5 seconds after the addition of D-GAP to pre-formed [C2β-13C]-LThDP.

Figure 8

a) Formation of 1′, 4′–iminopyrimidyl-LThDP (318 nm) from pyruvate (10-200 μM) by R420A DXP synthase (29.6 μM) at 5 °C; b) Pre-steady state analysis of LThDP formation and decarboxylation under single turnover conditions on R420A (30 μM) with pyruvate (15 μM), in the absence of D-GAP at 6 °C. Data were fitted to equation 3 (see text); c) Pre-steady state analysis of LThDP decarboxylation (k2) and resynthesis (k1) on R420A (30 μM was pre-mixed with 150 μM pyruvate to form LThDP) in the presence of 1 mM D-GAP at 6 °C (inset) fitting the expansion of early behavior to equation 4.

Figure 9

a-b) Pyruvate binding analysis by intrinsic protein fluorescence of Y392F DXP synthase; c) Formation of 1′, 4′–iminopyrimidyl-LThDP (313 nm) at 5 °C from pyruvate (30-500 .M) by Y392F DXP synthase (29.9 .M) at 5 °C and the corresponding binding curve (d); e) Pre-steady state analysis of LThDP formation and decarboxylation under single turnover conditions on Y392F (35 .M) with pyruvate (17.5 .M), in the absence of D-GAP at 6 °C. Data were fit to equation 2 (see text); f) Pre-steady state analysis of LThDP decarboxylation (k2) and (k1) re-synthesis on Y392F (27 .M was pre-mixed with 205 .M pyruvate to form LThDP) in the presence of 100 .M D-GAP at 6 °C (inset) fitting the expansion of early behavior to equation 4.

Figure 10

Pre-steady state DXP formation from pyruvate and D-GAP by DXP synthase variants at 6 °C a) R478A; b) R420A; c) Y392F.

Figure 11

Catalytic cycle of DXP synthase with microscopic rate constants referred to in Table 2. The rate constants presented in Table 2 were obtained by assuming irreversible forward rate constants.

Figure 12

a) Pre-steady state analysis of LThDP decarboxylation (k2) and re-synthesis (k1) on wild type DXP synthase (47.5 %M of enzyme was pre-mixed with 500 %M pyruvate to form LThDP) in the presence of 30 mM D-glyceraldehyde at 8 °C (inset) fitting the expansion of the early behavior to equation 4. b) Pre-steady state DX product formation from pyruvate and D-glyceraldehyde by wild type DXP synthase at 8 °C. Data were fitted to equation 5.