Crystallization and phasing of alanine dehydrogenase from Archaeoglobus fulgidus.
Alanine dehydrogenase (AlaDH) from the hyperthermophilic archaeon Archaeoglobus fulgidus is a dimer of 35 kDa chains. The archaeal enzyme appears to represent a new class of AlaDH that is not homologous to bacterial AlaDH enzymes, but has close evolutionary links to the broad ornithine cyclodeaminase/micro-crystallin family, which includes human thyroid hormone binding protein, which has 30% sequence identity to the A. fulgidus gene. The enzyme has been cloned, shown to catalyze the NAD-dependent interconversion of alanine and pyruvate and crystallized in several forms. Although the purified protein crystallized readily under many conditions, most of the crystals diffracted weakly or not at all. One polymorph growing in space group P2(1)2(1)2(1) has non-crystallographic symmetry that becomes crystallographic, changing the space group to P2(1)2(1)2, upon binding iridium or samarium. Before and after derivatization, these crystals diffracted to 2.5 A using synchrotron radiation. Multiwavelength diffraction data were collected from the non-isomorphous iridium derivative, enabling structure determination.
Smith
Natasha
N
Biotechnology Division of the National Institute of Standards and Technology, Gaithersburg, MD 20899-8312, USA.
Mayhew
Martin
M
Robinson
Howard
H
Héroux
Annie
A
Charlton
David
D
Holden
Marcia J
MJ
Gallagher
D T
DT
D050788:Alanine Dehydrogenase; D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics; D019611:Archaeoglobus fulgidus / Q000201:enzymology* / Q000235:genetics; D003460:Crystallization; D018360:Crystallography, X-Ray; D019281:Dimerization; D007495:Iridium / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D012493:Samarium / Q000737:chemistry; D017356:Synchrotrons
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D012493:Samarium; D007495:Iridium; D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase