National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01GM065546
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01GM061068
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
P30GM103335
United States
National Science Foundation (NSF, United States)
DBI-1156692
United States
Citation
Journal: J Biol Chem / Year: 2016 Title: Structures of Proline Utilization A (PutA) Reveal the Fold and Functions of the Aldehyde Dehydrogenase Superfamily Domain of Unknown Function. Authors: Min Luo / Thameesha T Gamage / Benjamin W Arentson / Katherine N Schlasner / Donald F Becker / John J Tanner / 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 ...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.
Mass: 18.015 Da / Num. of mol.: 604 / Source method: isolated from a natural source / Formula: H2O
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Experimental details
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Experiment
Experiment
Method: X-RAY DIFFRACTION / Number of used crystals: 1
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Sample preparation
Crystal
Density Matthews: 2.73 Å3/Da / Density % sol: 54.99 %
Crystal grow
Temperature: 295 K / Method: vapor diffusion, sitting drop / pH: 7.8 Details: 0.1 M Hepes at pH 7.8, 0.2 M ammonium sulfate, 0.1 M sodium formate, 0.1 M MgCl2, and 22% (w/v) PEG 3350
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Data collection
Diffraction
Mean temperature: 100 K
Diffraction source
Source: SYNCHROTRON / Site: ALS / Beamline: 4.2.2 / Wavelength: 1 Å
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