Structures of Proline Utilization A (PutA) Reveal the Fold and Functions of the Aldehyde Dehydrogenase Superfamily Domain of Unknown Function

Abstract

Aldehyde dehydrogenases (ALDHs) catalyze the NAD(P)-dependent oxidation of aldehydes to carboxylic acids and are important for metabolism and detoxification. Although the ALDH superfamily fold is well established, some ALDHs contain an uncharacterized domain of unknown function (DUF) near the C terminus of the polypeptide chain. Herein, we report the first structure of a protein containing the ALDH superfamily DUF. Proline utilization A from Sinorhizobium meliloti (SmPutA) is a 1233-residue bifunctional enzyme that contains the DUF in addition to proline dehydrogenase and l-glutamate-γ-semialdehyde dehydrogenase catalytic modules. Structures of SmPutA with a proline analog bound to the proline dehydrogenase site and NAD bound to the ALDH site were determined in two space groups at 1.7-1.9 Å resolution. The DUF consists of a Rossmann dinucleotide-binding fold fused to a three-stranded β-flap. The Rossmann domain resembles the classic ALDH superfamily NAD-binding domain, whereas the flap is strikingly similar to the ALDH superfamily dimerization domain. Paradoxically, neither structural element performs its implied function. Electron density maps show that NAD does not bind to the DUF Rossmann fold, and small-angle X-ray scattering reveals a novel dimer that has never been seen in the ALDH superfamily. The structure suggests that the DUF is an adapter domain that stabilizes the aldehyde substrate binding loop and seals the substrate-channeling tunnel via tertiary structural interactions that mimic the quaternary structural interactions found in non-DUF PutAs. Kinetic data for SmPutA indicate a substrate-channeling mechanism, in agreement with previous studies of other PutAs.

Proline utilization system is required for infection by the pathogenic α-proteobacterium Brucella abortus

“…The L-proline dehydrogenase activity of rPutA was determined as described previously, but with modifications [25,26]. The reaction was carried out at room temperature (22 C) in a solution containing 50 mM potassium phosphate buffer (pH 7.5), 25 mM NaCl (pH 7.5), 200 µM NAD + , 240 µM coenzyme Q1 (CoQ1), 20 mM L-proline, 1 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 1 mM 5-methylphenazinium methyl sulfate (PMS) and 0.072 µM rPutA.…”

“…The reaction was initiated by the addition of rPutA and monitored spectrophotometrically at 568 nm. L-Proline dehydrogenase reaction generated NADH [26], which reduced PMS, which in turn reduced MTT, producing formazan and causing a rise in absorbance at 568 nm [25].…”

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

“…Architecture type A PutAs are about 1000 residues long and have a minimal domain set consisting of N-terminal PRODH and C-terminal GSALDH modules. The type B PutAs have an additional 100–200 residue C-terminal domain, which was recently shown to be a structural domain having the aldehyde dehydrogenase superfamily (ALDHSF) fold [29, 30]. Type C PutAs contain both the C-terminal ALDHSF domain and an N-terminal ribbon-helix-helix (RHH) DNA-binding domain, the latter endowing these PutAs with transcriptional repressor functionality.…”

“…Structures of type B PutAs from Sinorhizobium meliloti (SmPutA) [30] and Corynebacterium freiburgense (CfPutA) [29] confirmed the conservation of the PutA fold and revealed the structure and functions of the C-terminal ALDHSF domain. Type B PutAs have 6 of the 7 domains seen in type A PutAs, plus the C-terminal domain, which is not found in type A PutAs (Fig.…”

“…1B). The C-terminal domain spans about 200 residues and consists of an α/β sub-domain fused to a β-flap [29, 30]. Curiously, the α/β sub-domain resembles the Rossmann fold found in all ALDHs, including the GSALDH module of PutA.…”