FUNCTIONAL MAPPING OF BRUCELLA ABORTUS CYCLIC GLUCAN SYNTHASE BY PENTAPEPTIDE SCANNING MUTAGENESIS

GUIDOLIN, L.S.; CIOCCHINI, A. E.; IÑÓN DE IANNINO, N. ; UGALDE, R. A.

Rosario, Santa Fe, Argentina

Congreso; XLII Reunion Anual de la Sociedad Argentina de Investigaciones en Bioquimica y Biologia Molecular (SAIB 2006); 2006

Sociedad Argentina de Investigaciones en Bioquimica y Biologia Molecular (SAIB)

B. abortus cyclic b-1,2-glucan synthase (Cgs) is a 320-kDa polytopic integral inner membrane protein responsible for the synthesis of the virulence factor cyclic b-1,2-glucan by a novel mechanism in which the enzyme itself acts as a protein intermediate. Cgs functions as an inverting processive b-1,2-autoglucosyltransferase and has the three-enzymatic activities required for synthesis of the cyclic glucan: initiation, elongation, and cyclization. To identify functionally important regions of Cgs, we used random pentapeptide insertion mutagenesis and assessed the function of the resulting mutants by a mobility assay. We analyzed 2464 Cgs mutants and mapped by sequencing the exact localization of the insertion in 330 of them. In this way, we built a functional map of Cgs from which we identified various domains required for overall function and delimited the minimal region of the protein required for the synthesis of cyclic glucan. Then, we characterized in vitro and in vivo the activity of several selected mutants. Particularly, we found that most of the pentapeptide insertions into the Cgs region between the residues 990 and 1547 inactivated the synthesis of cyclic glucan but the incorporation of glucose residues into the protein through the initiation and elongation reactions was not affected. Therefore, this region of the protein may be implicated in the cyclization reaction.