Structures of Proline Utilization A (PutA) Reveal the Fold and Functions of the Aldehyde Dehydrogenase Superfamily Domain of Unknown Function.
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.
From the Departments of Chemistry and.
Min
M
Luo
From the Departments of Chemistry and.
Thameesha T
TT
Gamage
the Department of Biochemistry and Redox Biology Center, University of Nebraska, Lincoln, Nebraska 68588.
Benjamin W
BW
Arentson
the Department of Biochemistry and Redox Biology Center, University of Nebraska, Lincoln, Nebraska 68588.
Katherine N
KN
Schlasner
the Department of Biochemistry and Redox Biology Center, University of Nebraska, Lincoln, Nebraska 68588.
Donald F
DF
Becker
From the Departments of Chemistry and tannerjj@missouri.edu.|Biochemistry, University of Missouri, Columbia, Missouri 65211, and.
John J
JJ
Tanner
D000444:Aldehyde Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009243:NAD; D000072417:Protein Domains; D017510:Protein Folding; D017433:Protein Structure, Secondary; D016962:Sinorhizobium meliloti / Q000201:enzymology* / Q000235:genetics; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D009243:NAD; D000444:Aldehyde Dehydrogenase
X-ray crystallography; aldehyde dehydrogenase superfamily; enzyme kinetics; flavoprotein; nicotinamide adenine dinucleotide (NAD); oligomerization; proline catabolism; proline utilization A; protein domain; small-angle X-ray scattering (SAXS)
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