Kinetic and structural characterization of tunnel-perturbing mutants in Bradyrhizobium japonicum proline utilization A

Abstract

Proline utilization A from Bradyrhizobium japonicum (BjPutA) is a bifunctional flavoenzyme that catalyzes the oxidation of proline to glutamate using fused proline dehydrogenase (PRODH) and Δ(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Recent crystal structures and kinetic data suggest an intramolecular channel connects the two active sites, promoting substrate channeling of the intermediate Δ(1)-pyrroline-5-carboxylate/glutamate-γ-semialdehyde (P5C/GSA). In this work, the structure of the channel was explored by inserting large side chain residues at four positions along the channel in BjPutA. Kinetic analysis of the different mutants revealed replacement of D779 with Tyr (D779Y) or Trp (D779W) significantly decreased the overall rate of the PRODH-P5CDH channeling reaction. X-ray crystal structures of D779Y and D779W revealed that the large side chains caused a constriction in the central section of the tunnel, thus likely impeding the travel of P5C/GSA in the channel. The D779Y and D779W mutants have PRODH activity similar to that of wild-type BjPutA but exhibit significantly lower P5CDH activity, suggesting that exogenous P5C/GSA enters the channel upstream of Asp779. Replacement of nearby Asp778 with Tyr (D778Y) did not impact BjPutA channeling activity. Consistent with the kinetic results, the X-ray crystal structure of D778Y shows that the main channel pathway is not impacted; however, an off-cavity pathway is closed off from the channel. These findings provide evidence that the off-cavity pathway is not essential for substrate channeling in BjPutA.

Scheme 1. Overall Reaction Catalyzed by Proline Utilization A (PutA)

Flavin-dependent proline dehydrogenase (PRODH) catalyzes the oxidation of proline to Δ¹-pyrroline-5-carboxylate (P5C) and reduction of respiratory quinones in the membrane (Mem). P5C undergoes a nonenzymatic hydrolysis, resulting in glutamate-γ-semialdehyde (GSA). GSA is oxidized to glutamate by P5C dehydrogenase (P5CDH) using an NAD⁺ cofactor.

Figure 1

Tunnel/cavity system of BjPutA. (A) BjPutA protomer with PRODH colored blue, P5CDH pink, and the oligomerization flap green. The FAD and NAD⁺ are shown as yellow and green sticks, respectively. Catalytic Cys792 of the P5CDH active site is indicated. The gray surface represents the predicted channeling pathway calculated with MOLE. Helices α5a and 770s (residues 773–785) are colored gold and cyan, respectively. We note that in a tetramer of BjPutA, the dimerization flap of one protomer covers the tunnel of the other protomer. (B) Details of the predicted channeling pathway. The predicted path from MOLE is shown as mesh. Models of P5C and GSA in the tunnel are shown for scale (green). (C) Another view of the tunnel/cavity system, with the predicted channeling tunnel calculated from MOLE shown as gray mesh and the off-pathway cavity calculated using VOIDOO shown as red mesh.

Figure 2

Channeling assays of wild-type BjPutA and its mutants. Assays were performed in 50 mM potassium phosphate (pH 7.5, 25 mM NaCl, 10 mM MgCl₂) with 0.187 μM BjPutA enzyme, 40 mM proline, 100 μM CoQ₁, and 200 μM NAD⁺.

Figure 3

Channeling assays with increasing concentrations of D779Y (A) and D779W (B). NADH formation was monitored using fluorescence by exciting at 340 nm and recording the emission at 460 nm. Assays were performed with wild-type BjPutA (0.187 μM) and increasing concentrations of mutants (0.187–1.87 μM) in 50 mM potassium phosphate (pH 7.5, 25 mM NaCl, 10 mM MgCl₂) containing 40 mM proline, 100 μM CoQ₁, and 200 μM NAD⁺.

Figure 4

Binding of NAD⁺ to BjPutA. (A) Wild-type BjPutA (0.25 μM) was titrated with increasing concentrations of NAD⁺ (0–20 μM) in 50 mM potassium phosphate buffer (pH 7.5). The inset is a plot of the change in tryptophan fluorescence vs [NAD⁺] fit to a single-site binding isotherm. A K_d value of 0.60 ± 0.04 μM was estimated for the NAD⁺–BjPutA complex. (B) ITC analysis of binding of NAD⁺ to wild-type BjPutA. The top panel shows the raw data of wild-type BjPutA (23.4 μM) titrated with increasing amounts of NAD⁺ in 50 mM Tris buffer (pH 7.5). The bottom panel shows the integration of the titration data. The binding of NAD⁺ to BjPutA is shown to be exothermic, and a best fit of the data to a single-site binding isotherm yielded a K_d of 1.5 ± 0.2 μM.

Figure 5

Single-turnover rapid-reaction kinetic data for wild-type BjPutA and mutant D779Y. (A) Wild-type BjPutA (21.3 μM) and (B) BjPutA mutant D779Y (17.9 μM) were incubated with 100 μM NAD⁺ and rapidly mixed with 40 mM proline (all concentrations reported as final) and monitored by stopped-flow multiwavelength absorption (300–700 nm). Insets showing FAD (451 nm) and NAD⁺ (340 nm) reduction vs time fit to a single-exponential equation to obtain the observed rate constant (k_obs) of FAD and NAD⁺ reduction. Note that the inset in panel B is on a longer time scale.

Figure 6

Electron density maps and local conformational changes. (A) Electron density map for D778Y. (B) Electron density map for D779Y. (C) Electron density map for D779W. (D) Superposition of BjPutA (gray), D778Y (gold), D779Y (cyan), and D779W (magenta). The cages in panels A–C represent simulated annealing σ_A-weighted F₀ - F_c omit maps contoured at 2.5σ.

Figure 7

Invasion of the off-pathway cavity by Tyr778 in D778Y. The gray cylinder represents the channeling pathway calculated from the wild-type BjPutA structure (PDB entry 3HAZ) using MOLE, and the view is from the P5CDH active site looking through the tunnel toward the PRODH site. The red mesh represents the off-pathway cavity of wild-type BjPutA calculated using VOIDOO, while the blue surface represents the residual off-pathway cavity of D778Y, also calculated with VOIDOO.

Figure 8

Constriction of the channeling tunnel by Tyr779 in D779Y. (A) The gray cylinder represents the channeling pathway calculated from the wild-type BjPutA structure (PDB entry 3HAZ) using MOLE, and the view is from the P5CDH active site looking through the tunnel toward the PRODH site. (B) Comparison of the predicted channeling pathway of wild-type BjPutA (gray surface) and D779Y (red mesh).

Figure 9

Constriction of the channeling tunnel by Trp779 in D779W. (A) The gray cylinder represents the channeling pathway calculated from the wild-type BjPutA structure (PDB entry 3HAZ) using MOLE, and the view is from the P5CDH active site looking through the tunnel toward the PRODH site. (B) Comparison of the predicted channeling pathway of wild-type BjPutA (gray surface) and D779W (red mesh).