Three-dimensional structure of C. Glutamicum DAP dehydrogenase

G. Scapin, S. Reddy, J. C. Sacchettini, John S. Blanchard

Research output: Contribution to journalArticle

Abstract

Diaminopimelate dehydrogenase (DAP-DH) catalyzes the single step conversion of L-2-amino-6-ketopimeIate to meso-DAP, the direct precursor of L-lysine in the bacterial lysine synthetic pathway. The enzyme catalyzes the oxidative deamination of an amino acid of D configuration, and must additionally distinguish between two chiral centers on the same symmetric substrate, we have therefore initiated crystallographic studies to probe the structural basis for this stereospecific discrimination. DAP-DH has been cloned, expressed in E.coli and purified to homogeneity using standard biochemical procedures The crystalline structure of the complex DAPDH-NADP(+) has been solved using multiple isomorphous replacement procedures and non-crystallographic symmetry averaging. The resulting model has been refined against 2.4 A diffraction data to a crystallographic R-factor of 15.1%. DAP-DH is a homodimer of structurally not-identical subunits, Each subunit is composed of two domains, with a deep cleft in between. The N-terminal domain contains a modified dinucleotide binding domain or Rossman fold (6 central β-strands in a 213456 topology, surrounded by 4 α-helices). The C-terminal domain is composed of 6 β-strands and 5 α-helices. The dinier interface involves mainly residue from the C-terminal portion of the protein. The relative positions of the two domains in the two monomers are different, possibly due to different lattice contacts. The nucleotide is bound in an extended conformation across the C-terminal portion of the β-sheet of the Rossman fold, with its C4 facing the cleft between the two domains. The structure of DAP-DH closely resembles the structure of dihydrodipicolinate reductase, the enzyme immediately preceding DAP-DH in the DAP/lysine biosynthetic pathway.

Original languageEnglish (US)
JournalFASEB Journal
Volume10
Issue number6
StatePublished - 1996

Fingerprint

diaminopimelate dehydrogenase
lysine
Oxidoreductases
Lysine
deamination
Dihydrodipicolinate Reductase
R388
enzymes
crystal structure
NADP (coenzyme)
topology
biochemical pathways
nucleotides
Deamination
Escherichia coli
Biosynthetic Pathways
Enzymes
amino acids
NADP
Conformations

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Three-dimensional structure of C. Glutamicum DAP dehydrogenase. / Scapin, G.; Reddy, S.; Sacchettini, J. C.; Blanchard, John S.

In: FASEB Journal, Vol. 10, No. 6, 1996.

Research output: Contribution to journalArticle

Scapin, G, Reddy, S, Sacchettini, JC & Blanchard, JS 1996, 'Three-dimensional structure of C. Glutamicum DAP dehydrogenase', FASEB Journal, vol. 10, no. 6.
Scapin, G. ; Reddy, S. ; Sacchettini, J. C. ; Blanchard, John S. / Three-dimensional structure of C. Glutamicum DAP dehydrogenase. In: FASEB Journal. 1996 ; Vol. 10, No. 6.
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N2 - Diaminopimelate dehydrogenase (DAP-DH) catalyzes the single step conversion of L-2-amino-6-ketopimeIate to meso-DAP, the direct precursor of L-lysine in the bacterial lysine synthetic pathway. The enzyme catalyzes the oxidative deamination of an amino acid of D configuration, and must additionally distinguish between two chiral centers on the same symmetric substrate, we have therefore initiated crystallographic studies to probe the structural basis for this stereospecific discrimination. DAP-DH has been cloned, expressed in E.coli and purified to homogeneity using standard biochemical procedures The crystalline structure of the complex DAPDH-NADP(+) has been solved using multiple isomorphous replacement procedures and non-crystallographic symmetry averaging. The resulting model has been refined against 2.4 A diffraction data to a crystallographic R-factor of 15.1%. DAP-DH is a homodimer of structurally not-identical subunits, Each subunit is composed of two domains, with a deep cleft in between. The N-terminal domain contains a modified dinucleotide binding domain or Rossman fold (6 central β-strands in a 213456 topology, surrounded by 4 α-helices). The C-terminal domain is composed of 6 β-strands and 5 α-helices. The dinier interface involves mainly residue from the C-terminal portion of the protein. The relative positions of the two domains in the two monomers are different, possibly due to different lattice contacts. The nucleotide is bound in an extended conformation across the C-terminal portion of the β-sheet of the Rossman fold, with its C4 facing the cleft between the two domains. The structure of DAP-DH closely resembles the structure of dihydrodipicolinate reductase, the enzyme immediately preceding DAP-DH in the DAP/lysine biosynthetic pathway.

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