Crystal Structure of NAD-dependent Peptoniphilus Asaccharolyticus Glutamate Dehydrogenase Reveals Determinants of Cofactor Specificity
Citation:
Tânia Oliveira, Santosh Panjikar, John B. Carrigan, Muaawia Hamza, Paul C. Engel, Amir R. Khan, Crystal Structure of NAD-dependent Peptoniphilus Asaccharolyticus Glutamate Dehydrogenase Reveals Determinants of Cofactor Specificity, Journal of Structural Biology, 177, 2, 2012, 543 552Download Item:
Crystal Structure of NAD-dependent Peptoniphilus Asaccharolyticus Glutamate Dehydrogenase Reveals Determinants of Cofactor Specificity.pdf (Published (author's copy) - Peer Reviewed) 3.184Mb
Abstract:
Glutamate dehydrogenases (EC 1.4.1.2?4) catalyse the oxidative deamination of l-glutamate to ?-ketoglutarate using NAD(P) as a cofactor. The bacterial enzymes are hexamers and each polypeptide consists of an N-terminal substrate-binding (Domain I) followed by a C-terminal cofactor-binding segment (Domain II). The reaction takes place at the junction of the two domains, which move as rigid bodies and are presumed to narrow the cleft during catalysis. Distinct signature sequences in the nucleotide-binding domain have been linked to NAD vs. NADP specificity, but they are not unambiguous predictors of cofactor preferences. Here, we have determined the crystal structure of NAD?specific Peptoniphilus asaccharolyticus glutamate dehydrogenase in the apo state. The poor quality of native crystals was resolved by derivatization with selenomethionine, and the structure was solved by single-wavelength anomalous diffraction methods. The structure reveals an open catalytic cleft in the absence of substrate and cofactor. Modeling of NAD+ in domain II suggests that a hydrophobic pocket and polar residues contribute to nucleotide specificity. Mutagenesis and isothermal titration calorimetry studies of a critical glutamate at the P7 position of the core fingerprint confirms its role in NAD binding. Finally, the cofactor binding site is compared with bacterial and mammalian enzymes to understand how the amino acid sequences and three-dimensional structures may distinguish between NAD vs. NADP recognition.
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Grant Number
Science Foundation Ireland (SFI)
03/IN.3/B371
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http://people.tcd.ie/amirrafkDescription:
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Author: KHAN, AMIR
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Journal of Structural Biology177
2
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