Interactions at the 2 and 5 positions of 5-phosphoribosyl pyrophosphate are essential in Salmonella typhimurium quinolinate phosphoribosyltransferase

Abstract

Quinolinate phosphoribosyltransferase (QAPRTase, EC 2.4.2.19) catalyzes an unusual phosphoribosyl transfer that is linked to a decarboxylation reaction to form the NAD precursor nicotinate mononucleotide, carbon dioxide, and pyrophosphate from quinolinic acid (QA) and 5-phosphoribosyl 1-pyrophosphate (PRPP). Structural studies and sequence similarities with other PRTases have implicated Glu214, Asp235, Lys153, and Lys284 in contributing to catalysis through direct interaction with PRPP. The four residues were substituted by site-directed mutagenesis. A nadC deletant form of BL21DE3 was created to eliminate trace contamination by chromosomal QAPRTase. The mutant enzymes were readily purified and retained their dimeric aggregation state on gel filtration. Substitution of Lys153 with Ala resulted in an inactive enzyme, indicating its essential nature. Mutation of Glu214 to Ala or Asp caused at least a 4000-fold reduction in k(cat), with 10-fold increases in K(m) and K(D) values for PRPP. However, mutation of Glu214 to Gln had only modest effects on ligand binding and catalysis. pH profiles indicated that the deprotonated form of a residue with pK(a) of 6.9 is essential for catalysis. The WT-like pH profile of the E214Q mutant indicated that Glu214 is not that residue. Mutation of Asp235 to Ala did not affect ligand binding or catalysis. Mutation of Lys284 to Ala decreased k(cat) by 30-fold and increased K(m) and K(D) values for PRPP by 80-fold and at least 20-fold, respectively. The study suggests that Lys153 is necessary for catalysis and important for PRPP binding, Glu214 provides a hydrogen bond necessary for catalysis but does not act as a base or electrostatically to stabilize the transition state, Lys284 is involved in PRPP binding, and Asp235 is not essential.

Figure 1

Active site of M. tuberculosis QAPRTase•PA•PRPCP•2Mn²⁺ Michaelis complex. The image shows the interactions of Lys153, Glu214, Asp235 and His284 (corresponding to Lys284 of S. typhimurium) with PRPCP-Mn²⁺ complex. Hydrogen bonds are shown as dashed lines and the water molecule involved in coordinating one of the Mn²⁺ is represented by a red sphere. Carbon atoms are colored green, nitrogen are blue, oxygen are red and phosphorous in orange and Mn²⁺ in purple. PA and the second Mn²⁺ coordinated by two oxygen of the pyrophosphate group of PRPCP are not shown. All distances shown in black are given in Å. The image was generated using PyMOL (Delano Scientific) and PDB entry 1QPR.

Figure 2

Partial sequence alignment of QAPRTase from various organisms. 10 of the 230 sequences aligned as described in Methods are displayed. Sequences are: SALTY, Salmonella typhimurium LT2; ECOLI, Escherichia coli K-12; MYCTU, Mycobacterium tuberculosis H37Rv; HUMAN, Homo sapiens; SYNFM, Syntrophobacter fumaroxidans; MESSB, Mesorhizobium sp. BNC1; RHOPS, Rhodopseudomonas palustris BisB5; ERYLH, Erythrobacter litoralis; SYNS3, Synechococcus sp. CC9311; METST, Methanosphaera stadtmanae. Residue numbering at top is from the mature S. typhimurium protein sequence. Highly conserved Glu214 and Asp235 residues are indicated in bold and marked with asterisks. Moderately conserved Lys284 is marked with a plus sign. A full display and sequence sources is found in Supporting Information.

Figure 3

Scatchard plot for the binding of [³H]QA to mutant enzymes. E214A (●), E214D (□) and E214Q (○). The values of K_D were 45 µM, 20 µM and 50 µM respectively.

Figure 4

Scatchard plot for the binding of [¹⁴C]PRPP to mutant apo-enzymes and QAPRTase•PA binary complex. [¹⁴C]PRPP binding by E214A (□), E214D (○), E214Q (▽) and to the E214A•PA (■), E214D•PA (●), and E214Q•PA (▼) complexes. The K_D values are reported in Table 1.

Figure 5

Pre-steady state kinetics of [³H]NAMN formation by WT and mutant enzymes. Rapid mixing was performed as described in Methods. (A) The k_cat for WT(●) reaction was 0.6 s⁻¹ and that for E214Q (○) was 0.5 s⁻¹. (B) The k_cat values for E214A(■), E214D (□) were 1.6×10⁻⁵ s⁻¹. (C) k_cat for K284A was 0.03 s⁻¹.

Figure 5

Pre-steady state kinetics of [³H]NAMN formation by WT and mutant enzymes. Rapid mixing was performed as described in Methods. (A) The k_cat for WT(●) reaction was 0.6 s⁻¹ and that for E214Q (○) was 0.5 s⁻¹. (B) The k_cat values for E214A(■), E214D (□) were 1.6×10⁻⁵ s⁻¹. (C) k_cat for K284A was 0.03 s⁻¹.

Figure 6

Stopped-flow studies with WT and E214Q QAPRTases. (A) Reaction traces obtained from Stopped-flow fluorescence analysis. Time dependence decay of intrinsic WT QAPRTase fluorescence upon mixing the QAPRTase•QA complex (6 µM QAPRTase incubated with 300 µM QA) with various concentrations of PRPP at pH 7.2, 25 °C (λ_ex = 290 nm, λ_em = 340 nm). Final PRPP concentrations were (a) 0, (b) 10, (c) 25, (d) 50, (e) 100, (f) 150, (g) 200, (h) 500, (i) 350, (j) 700, (k) 1000, (l) 1300, (m) 1600 and (n) 2000 µM. The traces were fit to a single exponential decay function (eq I). (B) Dependence of the observed rate constants of data from Figure A on PRPP concentration for WT (●) and E214Q (○).

Figure 7

pH Dependence of log k_cat with WT (●), E214Q (○), E214A (*) and E214D (▲). Lines represent fits of the data to eq IV with pK_a = 6.92 (WT) and 6.75 (E214Q).

Scheme 1

The reaction catalyzed by QAPRTase

Scheme 2

Kinetics of PRPP binding