Crystallization of NAD+-dependent phenylalanine dehydrogenase from Nocardia sp239.
The NAD+-dependent phenylalanine dehydrogenase from Nocardia sp239 has been crystallized by the hanging-drop method of vapour diffusion using ammonium sulfate as the precipitant. Two crystal forms were obtained in the presence and absence of the enzyme substrates phenylpyruvic acid or phenylalanine and its coenzyme NADH. Crystals of the native protein belong to the hexagonal system, with the space group being one of the enantiomorphic pair P6122 or P6522. The cell dimensions are a = b = 111.0, c = 174.5 A, alpha = beta = 90 and gamma = 120 degrees. Crystals grown from the protein co-crystallized with its substrates all belong to the trigonal system, space group P3121 or P3221, with unit-cell dimensions of a = b = 88.1, c = 112.6 A, alpha = beta = 90 and gamma = 120 degrees. Preliminary protein-sequencing experiments have established that this enzyme is related to the octameric PheDH's which are members of the wider superfamily of amino-acid dehydrogenases. However, gel-filtration studies suggest that this enzyme is active as a monomer. The full determination of the three-dimensional structure of this phenylalanine dehydrogenase will add to the understanding of the molecular basis of the differential substrate specificity within this enzyme superfamily. In turn this will contribute to the rational design of an amino-acid dehydrogenase which could be used for the diagnosis of phenylketonuria and for the chiral synthesis of high-value pharmaceuticals.
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, England.
A
A
Pasquo
K L
KL
Britton
P J
PJ
Baker
G
G
Brearley
R J
RJ
Hinton
A J
AJ
Moir
T J
TJ
Stillman
D W
DW
Rice
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D003460:Crystallization; D018360:Crystallography, X-Ray; D006801:Humans; D008969:Molecular Sequence Data; D009615:Nocardia / Q000201:enzymology* / Q000235:genetics; D010661:Phenylketonurias / Q000175:diagnosis; D017386:Sequence Homology, Amino Acid
D000594:Amino Acid Oxidoreductases; C073215:phenylalanine dehydrogenase (NAD)