X-ray Structure of Lipoamide Dehydrogenase from Azotobacter vinelandii Determined by a Combination of Molecular and Isomorphous Replacement Techniques

A.J. Schierbeek, M.B.A. Swarte, B.W. Dijkstra, G. Vriend, R.J. Read, W.G.J. Hol, J. Drenth, C. Betzel

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The crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii has been determined by a combination of molecular replacement and isomorphous replacement techniques yielding eventually a good-quality 2.8 Å electron density map. Initially, the structure determination was attempted by molecular replacement procedures alone using a model of human glutathione reductase, which has 26% sequence identity with this bacterial dehydrogenase. The rotation function yielded the correct orientation of the model structure both when the glutathione reductase dimer and monomer were used as starting model. The translation function could not be solved, however. Consequently, data for two heavy-atom derivatives were collected using the Hamburg synchotron facilities. The derivatives had several sites in common, which was presumably a major reason why the electron density map obtained by isomorphous information alone was of poor quality. Application of solvent flattening procedures cleaned up the map considerably, however, showing clearly the outline of the lipoamide dehydrogenase dimer, which has a molecular weight of 100,000. Application of the “phased translation function”, which combines the phase information of both isomorphous and molecular replacement, led to an unambiguous determination of the position of the model structure in the lipoamide dehydrogenase unit cell. The non-crystallographic 2-fold axis of the dimer was optimized by several cycles of constrained-restrained least-squares refinement and subsequently used for phase improvement by 2-fold density averaging. After ten cycles at 3.5 Å, the resolution was gradually extended to 2.8 Å in another 140 cycles. The 2.8 Å electron density distribution obtained in this manner was of much improved quality and allowed building of an atomic model of A. vinelandii lipoamide dehydrogenase. It appears that in the orthorhombic crystals used each dimer is involved in contacts with eight surrounding dimers, leaving unexplained why the crystals are rather fragile. Contacts between subunits within one dimer, which are quite extensive, can be divided into two regions separated by a cavity. In one of the contact regions, the level of sequence identity with glutathione reductase is very low but it is quite high in the other. The folding of the polypeptide chain in each subunit is quite similar to that of glutathione reductase, as is the extended conformation of the co-enzyme FAD. The structure of A. vinelandii lipoamide dehydrogenase solved forms a starting point for investigating details of the catalytic mechanism as well as studying the interactions of this enzyme with its partners in the pyruvate dehydrogenase multi-enzyme complex.
Original languageEnglish
Pages (from-to)365-379
Number of pages15
JournalJournal of Molecular Biology
Issue number2
Publication statusPublished - 20-Mar-1989


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