title: 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.
authors.affiliation: Department of Chemistry, University of Missouri-Columbia , Columbia, Missouri 65211, United States.
authors.forename: Min
authors.initials: M
authors.lastname: Luo
authors.forename: Shelbi
authors.initials: S
authors.lastname: Christgen
authors.forename: Nikhilesh
authors.initials: N
authors.lastname: Sanyal
authors.forename: Benjamin W
authors.initials: BW
authors.lastname: Arentson
authors.forename: Donald F
authors.initials: DF
authors.lastname: Becker
authors.forename: John J
authors.initials: JJ
authors.lastname: Tanner
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020369:Bradyrhizobium / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D005798:Genes, Bacterial; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000235:genetics / Q000378:metabolism*; D053838:Scattering, Small Angle; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; C080683:PutA protein, Bacteria; D011994:Recombinant Proteins; D000444:Aldehyde Dehydrogenase