STRUCTURAL STUDY OF THE NADP DEPENDENT ALDEHYDE DEHYDROGENASE FROM STREPTOCOCCUS MUTANS
D. Cobessi1, F. Tete-Favier1, S. Marchal2, G. Branlant2 and A. Aubry1
1Groupe Biocristallographie, Laboratoire
de Cristallographie et de Modélisation des Matériaux Minéraux
et Biologiques, UPRESA CNRS 7036, B.P. 239, 54506
Vandoeuvre-les-Nancy, France.
email: cobessi@lcm3b.u-nancy.fr
2Laboratoire de Maturation des ARN et
Enzymologie Moléculaire, UMR 7567, 54506
Vandoeuvre-les-Nancy, France.
Keywords: Crystal structure, Enzyme, Aldehyde
Dehydrogenase
The Aldehyde Dehydrogenases (ALDH) are a family of enzymes which catalyzes the oxidation of adehydes to their corresponding carboxylic acids. This family can be divided in three classes on the basis of their amino-acids sequences. The class 1 and the class 2 are tetrameric ALDHs and they share 70% sequence identity. The class 3 enzymes are dimeric and share 30% sequence identity with the two formers [1].
The Aldehyde Dehydrogenase of Streptococcus mutans catalyzes the irreversible oxidation of the glyceraldehyde-3-phosphate to 3-phosphoglycerate with reduction of NADP to NADPH [2,3]. This tetrameric enzyme of 200 kD shares 30% sequence identity with other ALDHs. Last year, the three first crystal structures of ALDH complexed with NAD were solved: one is dimeric and the others are tetrameric [4,5,6]. These three structures have a similar assembling of the catalytic domain and of the Rossman fold. The analysis of these structures reveals a new type of interactions between the Rossman fold and the NAD [4,5].
We have crystallized two forms of the ALDH (form1 and form2) from Streptococcus mutans in several conditions for the apo and holoenzyme complexed to NADP and we have collected the data to 1.8 A at LURE for the apoenzyme of form2 (Rsym: 6.1%, completness: 95%) and the data to 2.4 A at the laboratory for the form1 (apo and holoenzyme). The data for the holoenzyme of form2 have been collected to 2.6 A at ESRF.
The structure of apoenzyme of form2 was solved by molecular replacement (C=53.7, R=46.5) using AMoRe [7] with the model of the ALDH from cod liver [6] in which more than 300 amino-acids to 475 per monomer have been mutated in alanine.
At present, after several cycles of rebuilding and refinement at 1.8 A [8], the free R factor is 26.7% and the R factor is 23.5% for all data. The refinement is under progress.
At first glance, the overall folding of the protein is the same compared with the other enzymes of the family and the differences occur mainly in the loops and by the nature of amino-acids side chains.