Ketosteroid Isomerase (KSI) performs an important biological function in the metabolism of many bacteria by degrading steroids. Pseudomonas putida KSI (PpKSI) is strikingly efficient and selective. Three putative KSIs identified from Structural Genomics were analyzed by THEMATICS (THEoretical Microscopic Anomalous Titration Curve Shapes) and POOL (Partial Order Optimum Likelihood) and then characterized in vitro to determine KSI activity. These computational tools should allow us to predict whether each putative KSI does or does not have KSI activity. A putative KSI from Mycobacterium tuberculosis (MtKSI) was predicted to have ketosteroid isomerase activity, with putative KSIs from Mesorhizobium loti (MlKSI) and Pectobacterium atrosepticum (PaKSI) predicted to have no ketosteroid isomerase activity. Biochemical experiments reveal that the putative M. tuberculosis KSI does possess KSI activity, although with reduced efficiency compared to PpKSI. Proteins predicted to be misannotated, MlKSI and PaKSI, exhibited no detectable KSI activity. æWe engineered the MtKSI active site to resemble more closely that of PpKSI, and found that most of these mutations alone or in tandem significantly lowered rather than increased activity. Variants S16Y, F111D, S16Y/F64Y, S16Y/F111D, F64Y/F111D, and S16Y/F64Y/F111D were essentially inactive. Variant F64Y retained catalytic power, albeit no more than the wild-type enzyme. THEMATICS and POOL successfully differentiated between proteins that did and did not possess ketosteroid isomerase activity in Structural Genomics proteins assigned with putative functions. Although the active sites of MtKSI and PpKSI are similar, our attempts to increase the catalytic efficiency by creating a more PpKSI-like active site of MtKSI were not successful.