From Protein Structure to Function with Bioinformatics.pdf

11 Function Predictions of Structural Genomics Results 281The third case involved a protein (YjjX) for which the function could not beinferred from genomic location or sequence motifs. In this example the structureshowed a similar fold to a number of nucleotide binding proteins (including theMJ0226 protein discussed previously in the paper by Kim et al. 2003). Closerinspection of the active sites of YjjX and these structural matches identified significantsimilarity, with a number of residues either conserved or substituted withfunctionally equivalent residues. Further biochemical analysis identified the YjjXprotein as a novel ITPase/XTPase that acts as a housekeeping enzyme in E. coliduring oxidative stress to prevent the accumulation and subsequent incorporationinto nucleic acids of non-canonical nucleotides.The fourth case differed from the others in that it involved annotation of a proteinwithin a superfamily. Here the YhhW protein (previously annotated as a memberof the cupin superfamily) was structurally determined and, as anticipated,showed a similar core fold to known cupins with sequence suggesting the closestrelationship with the pirins. The broad range of functions within the cupin superfamilyprevented direct annotation from the global structural similarities so theauthors looked to local similarities in the molecular surfaces of the proteins. Theidentification of a deep charged pocket next to a metal binding site in YhhW andone of its homologues hPirin, suggested a possible active site. Inspection of thispocket showed significant similarity to that of quercetin 2,3-dioxygenase whichwas lent additional support through successful manual docking of quercetin to thepirin homologues. The quercetin 2,3-dioxygenase activity was confirmed by biochemicalassay and was the first enzymatic activity determined for any members ofthe pirin family. This example also illustrates the problems faced when dealing withlarge protein superfamilies. Often the global similarities such as the fold are insufficientto narrow down the function and more detailed local analysis is needed.The final example in this study is of another member of the cupin superfamily,the z3393 gene product from E. coli. Its sequence shows that it is more closelyrelated to the gentisate 1,2-dioxygenases than to other cupins. Global structural andlocal molecular surface comparisons lent support to the prediction of gentisate1,2-dioxygenase activity. The authors expect the structure of z3393 to aid futuremechanistic studies of the enzyme and to further understanding of how the gentisateoperon may be associated with pathogenic strains of E. coli.11.3 Some Specific ExamplesAlthough there are relatively few large-scale studies, there are a large number ofinteresting individual structures, published by the various structural genomics consortiaor in collaboration with them, where the structure has been vital for someelement of functional characterisation. One such example is that of Tm0936 fromThermotoga maritima (deposited as PDB entry 2plm) which was solved by a non-SG lab using clones provided by the JCSG. This protein had been deposited as aprotein of unknown function but is known to belong to the amidohydrolase family