Updated project metadata. Recent laboratory evolution studies have demonstrated that parallel evolutionary trajectories can lead to genetically distinct enzymes with high activity towards a non-preferred substrate. However, it is unknown whether such genetically distinct enzymes have convergent conformational dynamics and mechanistic features. To address this question, we used as a model the Homo sapiens kynureninase (HsKYNase), which is of great interest for cancer immunotherapy. Earlier, we reported on a highly unusual evolutionary trajectory that led to the isolation of HsKYNase_66, having a 410-fold increase in the kcat/KM for kynurenine (KYN) and reverse substrate selectivity relative to the parental wild-type enzyme. Here, by following a completely different evolutionary trajectory we generated a genetically distinct variant, HsKYNase_93D9, that exhibits KYN catalytic activity comparable to that of HsKYNase_66, but instead it is a “generalist” that accepts 3’-hydroxykynurenine (OH-KYN) with the same proficiency. Pre-steady-state kinetic analysis revealed that whereas the evolution of HsKYNase_66 was accompanied by a change in the rate-determining step of the reaction, HsKYNase_93D9 retained the same catalytic mechanism as the parental OH-KYN-preferring human enzyme. HDX-MS revealed that the conformational dynamics of the two enzymes during catalysis are markedly different and distinct from ortholog procaryotic enzymes with high KYN activity. Our work provides a mechanistic framework for understanding the relationship betweenevolutionary mechanisms and phenotypic traits of evolved generalist and specialist enzyme species.