Evidence that the C-terminal domain of a type B PutA protein contributes to aldehyde dehydrogenase activity and substrate channeling

Abstract

Proline utilization A (PutA) is a bifunctional enzyme that catalyzes the oxidation of proline to glutamate. Structures of type A PutAs have revealed the catalytic core consisting of proline dehydrogenase (PRODH) and Δ(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) modules connected by a substrate-channeling tunnel. Type B PutAs also have a C-terminal domain of unknown function (CTDUF) that is absent in type A PutAs. Small-angle X-ray scattering (SAXS), mutagenesis, and kinetics are used to determine the contributions of this domain to PutA structure and function. The 1127-residue Rhodobacter capsulatus PutA (RcPutA) is used as a representative CTDUF-containing type B PutA. The reaction progress curve for the coupled PRODH-P5CDH activity of RcPutA does not exhibit a time lag, implying a substrate channeling mechanism. RcPutA is monomeric in solution, which is unprecedented for PutAs. SAXS rigid body modeling with target-decoy validation is used to build a model of RcPutA. On the basis of homology to aldehyde dehydrogenases (ALDHs), the CTDUF is predicted to consist of a β-hairpin fused to a noncatalytic Rossmann fold domain. The predicted tertiary structural interactions of the CTDUF resemble the quaternary structural interactions in the type A PutA dimer interface. The model is tested by mutagenesis of the dimerization hairpin of a type A PutA and the CTDUF hairpin of RcPutA. Similar functional phenotypes are observed in the two sets of variants, supporting the hypothesis that the CTDUF mimics the type A PutA dimer interface. These results suggest annotation of the CTDUF as an ALDH superfamily domain that facilitates P5CDH activity and substrate channeling by stabilizing the aldehyde-binding site and sealing the substrate-channeling tunnel from the bulk medium.

Figure 1

PutA reactions and domain architectures. (A) Reactions catalyzed by PutA. (B) Domain diagrams representing the three types of PutA domain architectures.

Figure 2

Structure of BjPutA. (A) Protomer structure emphasizing the arrangement of domains. The surface represents the substrate-channeling tunnel calculated using Mole. (B) BjPutA dimer. The two protomers are colored according to the legend in panel A. The surfaces represent the substrate-channeling tunnels. (C) Close-up view of the dimer interface, emphasizing how the dimerization flap covers the substrate-channeling tunnel. (D) Interactions between the oligomerization domain (orange) and the GSA anchor loop of the P5CDH catalytic domain (cyan) in BjPutA. The locations of NAD⁺ and glutamate were inferred from the structures of BjPutA–NAD⁺ (PDB entry 3HAZ(5)) and P5CDH–glutamate (PDB entry 3V9K(45)) complexes. Residue numbers of RcPutA are listed in parentheses. The surface represents the substrate-channeling tunnel.

Figure 3

Kinetic analysis of the coupled PRODH–P5CDH reaction of RcPutA and BjPutA. (A) Steady-state progress curves of the production of NADH from proline by wild-type RcPutA (black circles), RcPutA1–1116 (red circles), and an equimolar mixture of RcPutA monofunctional variants R454M and C791A (green circles). (B) Steady-state progress curves of the production of NADH from proline by wild-type BjPutA (black circles), BjPutA1–986 (red circles), and an equimolar mixture of BjPutA monofunctional variants R456M and C792A (green circles). The dashed lines show the linear extrapolation used to estimate the lag time for each reaction.

Figure 4

SAXS analysis of RcPutA. (A) Scattering curve and Guinier analysis (inset). The Guinier plot spans the qR_g range of 0.364–1.29. (B) Pair distribution function. (C) Kratky plot and Porod–Debye plot (inset).

Figure 5

Determination of the molecular mass of RcPutA using MALS. The red curve represents the light scattering response measured at 90°. The black curve represents the response of the refractive index detector. The blue curve shows the derived molecular mass.

Figure 6

Shape reconstruction of RcPutA. (A) Shape reconstruction from GASBOR. The surface represents the averaged and filtered volume from 50 independent GASBOR calculations. The normalized spatial discrepancy is 1.02 ± 0.2. (B) Superposition of the GASBOR shape with a representative model from SAXS rigid body modeling [χ = 1.5, and rmsd = 1.0 (which corresponds to the green curve in Figure 8A)]. The catalytic core is colored red. The CTDUF with the C-terminal peptide composite model is colored blue. The spheres represent dummy residues linking the catalytic core and CTDUF.

Figure 7

Local sequence alignment of BjPutA residues 622–756 and the CTDUF of RcPutA. A homology model of the RcPutA CTDUF based on this alignment from the PHYRE2 server is shown, with identical residues highlighted in red.

Figure 8

Comparison of the experimental and theoretical SAXS curves. (A) Comparison of the experimental SAXS curve with theoretical curves calculated from the catalytic core model (orange) and three representative models of RcPutA from rigid body modeling. (B) Rigid body model of RcPutA that was used to calculate the green curve in panel A (χ = 1.5, and rmsd = 1.0).

Figure 9

SAXS rigid body modeling results from CORAL set 1. (A) Scatter plot of the clash penalty vs χ for the 160 poses of CORAL set 1 (red circles) and the 320 decoy poses (blue squares). The green oval encloses the high-confidence cluster of 78 poses, which is separated from the decoy poses. (B) Scatter plot of rmsd from the BjPutA dimer interface vs χ. The green circles represent the high-confidence cluster of poses from panel A. (C) Seventy-eight poses of the high-confidence cluster. The catalytic core is colored red. The β-hairpin and abbreviated Rossmann fold of the CTDUF are colored orange and blue, respectively.

Figure 10

SAXS rigid body modeling results from CORAL set 2. (A) Scatter plot of the clash penalty vs χ for the 480 poses of CORAL set 2 (red circles) and the 320 decoy poses (blue squares). The green oval encloses the high-confidence cluster of 253 poses, which is separated from the decoy poses. (B) Scatter plot of rmsd from the BjPutA dimer interface vs χ. The green circles represent the high-confidence cluster of poses from panel A. (C) Two hundred fifty-three poses of the high-confidence cluster. The catalytic core is colored red, the β-hairpin orange, the Rossmann fold blue, and the conserved C-terminal motif green.