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 Δ1-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.

Structure, function, and mechanism of proline utilization A (PutA)

“…Deletion of the C-terminal ALDHSF domain abrogates GSALDH activity, which supports the hypothesis proposed by Luo et al [30, 37] that the β-flap of the C-terminal domain stabilizes the aldehyde binding site. Somewhat surprisingly, deletion of the C-terminal ALDHSF domain also greatly diminished the PRODH activity of CfPutA [29].…”

“…Steady-state kinetics data are consistent with substrate channeling for all PutAs studied to date, including BjPutA [31], GsPutA [32], SmPutA [30], RcPutA [37], CfPutA [29], EcPutA [52], and a chimeric PutA consisting of the RHH domain of EcPutA fused to RcPutA [62]. Transient time analysis data collected on BjPutA are typical for PutAs (Fig.…”

“…SAXS studies of the type B PutA from Rhodobacter capsulatus (RcPutA) indicated a monomeric protein in solution [37]. SAXS analysis of SmPutA showed the enzyme forms a concentration-dependent monomer-dimer equilibrium [30].…”

“…Surprisingly, RcPutA, a bifunctional class 1B PutA, also displays some aspects of switching, such as proline-induced membrane binding [62]. Thus, the C-terminal ALDHSF domain of RcPutA is not only essential for coupling PRODH/GSALDH activities and facilitating substrate channeling, but also enables RcPutA to toggle between soluble and membrane bound forms [37, 62]. The physiological importance of this for type B PutAs is not apparent, but it seems possible that some essential elements of functional switching are "hard-wired" in the PRODH and C-terminal ALDHSF domains of type B and C PutAs.…”

Discovery of the Membrane Binding Domain in Trifunctional Proline Utilization A

“…36 The steady-state parameters K m and k cat for proline were determined as previously described using Ec PutA protein (0.01–0.3 μ M) and L-proline (0–700 mM) while keeping DCPIP fixed at 75 μ M. Assays were performed in 20 mM Tris-HCl buffer (pH 8.0) with 10% glycerol at 23 °C.…”