The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase from Haemophilus influenzae contains two active-site histidine residues

The catalytic and structural properties of the H67A and H349A dapE -encoded N -succinyl- l , l -diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. On the basis of sequence alignment with the carboxypeptidase from Pseudomonas sp. strain RS-16, both H67 and H349 we...

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Bibliographic Details
Published in:	Journal of biological inorganic chemistry Vol. 14; no. 1; pp. 1 - 10
Main Authors:	Gillner, Danuta M., Bienvenue, David L., Nocek, Boguslaw P., Joachimiak, Andrzej, Zachary, Vincentos, Bennett, Brian, Holz, Richard C.
Format:	Journal Article
Language:	English
Published:	Berlin/Heidelberg Springer-Verlag 01-01-2009
Subjects:	Amidohydrolases - chemistry Amidohydrolases - genetics Amidohydrolases - metabolism Amino Acid Sequence Biochemistry Biomedical and Life Sciences Catalytic Domain Cobalt - chemistry Cobalt - metabolism Diaminopimelic Acid - analogs & derivatives Diaminopimelic Acid - chemical synthesis Diaminopimelic Acid - chemistry Electron Spin Resonance Spectroscopy Haemophilus influenzae - enzymology Histidine - chemistry Histidine - genetics Histidine - metabolism Kinetics Life Sciences Microbiology Models, Molecular Molecular Sequence Data Mutagenesis, Site-Directed Original Paper Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - metabolism Sequence Alignment Spectrophotometry, Ultraviolet Zinc - chemistry Zinc - metabolism Homology model Biosynthesis Biomedicine Electron paramagnetic resonance Enzyme kinetics
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Summary:	The catalytic and structural properties of the H67A and H349A dapE -encoded N -succinyl- l , l -diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. On the basis of sequence alignment with the carboxypeptidase from Pseudomonas sp. strain RS-16, both H67 and H349 were predicted to be Zn(II) ligands. The H67A DapE enzyme exhibited a decreased catalytic efficiency (180-fold) compared with wild-type (WT) DapE towards N -succinyldiaminopimelic acid. No catalytic activity was observed for H349A under the experimental conditions used. The electronic paramagnetic resonance (EPR) and electronic absorption data indicate that the Co(II) ion bound to H349A-DapE is analogous to that of WT DapE after the addition of a single Co(II) ion. The addition of 1 equiv of Co(II) to H67A DapE provides spectra that are very different from those of the first Co(II) binding site of the WT enzyme, but that are similar to those of the second binding site. The EPR and electronic absorption data, in conjunction with the kinetic data, are consistent with the assignment of H67 and H349 as active-site metal ligands for the DapE from H. influenzae . Furthermore, the data suggest that H67 is a ligand in the first metal binding site, while H349 resides in the second metal binding site. A three-dimensional homology structure of the DapE from H. influenzae was generated using the X-ray crystal structure of the DapE from Neisseria meningitidis as a template and superimposed on the structure of the aminopeptidase from Aeromonas proteolytica (AAP). This homology structure confirms the assignment of H67 and H349 as active-site ligands. The superimposition of the homology model of DapE with the dizinc(II) structure of AAP indicates that within 4.0 Å of the Zn(II) binding sites of AAP all of the amino acid residues of DapE are nearly identical.
Bibliography:	Silesian University of Technology Loyola University Chicago Midwest Center for Structural Genomics Medical College of Wisconsin
ISSN:	0949-8257 1432-1327
DOI:	10.1007/s00775-008-0418-z