Structural Analysis of a Streptococcal Domain of Unknown Function(DUF1792)

        Serine-rich repeat proteins (SRRPs) belong to a growing family of bacterial adhesins required for biofilm formation and pathogenesis. Fap1 from Streptococcus parasanguinis is the first SRRP identified. A number of genes involved in Fap1 glycosylation have been characterized. Glycosyltransferases Gtf1, Gtf2 and Gtf3 catalyze the first and second steps of Fap1 glycosylation. A glycosyltransferase, GalT1, catalyzes the third step of Fap1 glycosylation.  At the N-terminus of GalT1, there is a domain of unknown function 1792 (DUF1792) that is highly conserved in bacteria and may constitute a broad protein superfamily, however its function is unknown.

Objective: Solve 3-D structure of DUF1792 to determine the function of the domain

Method:   The recombinant DUF1792 protein was purified via Ni²⁺ affinity chromatography and gel filtration. The hanging-drop vapor-diffusion method was used for the crystallization trials. The structure was determined by multiwavelength anomalous diffraction (MAD) utilizing Se atoms as the anomalous scatterers. The Gtf1-2-3 modified Fap1 was purified and used as a substrate to determine in vitro glycosyltransferase activity of DUF1792. An E. coli glycosylation system consisting of Fap1 substrate and respective glycosyltransferases was established to determine in vivo glycosylation activity.

Result:  A 3-D X-ray crystallographic structure of DUF1792 was determined at the resolution of 1.5 Å. The structure is composed of two N-terminal α/β/α sandwich domains, a C-terminal β/α/β Rossmann domain and a long-loop. In vitro and in vivo glycosyltransferase assays revealed that DUF1792 possesses glycosyltransferase activity, and the activity is conserved in other streptococci. A new metal binding motif required for glycosyltransferase activity was identified.

Conclusion: Since no sequence and structure homology analysis indicated DUF1792 as a glycosyltransferase, we concluded that DUF1792 represents a new family of glycosyltransferases.