Studies of bacterial catabolic enzymes: implications for the evolution of enzymes and metabolic pathways
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The origins of metabolic pathways and the evolution of the enzymes that comprise them have provoked intense debate and spawned a number of theories. The “patchwork” theory of Jensen, in which existing enzymes are combined to give new pathways, is one. Another emerging theme is that of “catalytic promiscuity,” the ability of an enzyme to catalyze a low-level activity that differs from its physiological function. Such an activity can be amplified through mutation(s) to yield a more efficient enzyme. These ideas are often used to explain the origins of “superfamilies,” which consist of enzymes that catalyze different reactions yet share sequence and/or structural homology. The catechol meta-fission pathway, a plasmid-encoded degradation pathway for simple aromatic compounds, is rich in enzyme chemistry and replete with structural and evolutionary diversity. 4-Oxalocrotonate tautomerase (4-OT), the best characterized enzyme in this pathway, is a member of the tautomerase superfamily. Two additional enzymes, YwhB, an enzyme of unknown function, and trans-3- chloroacrylic acid dehalogenase (CaaD) are also members. One defining feature of this superfamily is the conservation of an N-terminal proline which functions as a catalytic base. CaaD catalyzes dehalogenation via a hydration mechanism. Through site-directed mutagenesis, kinetic and pH rate analysis, and irreversible inhibition with 3-halopropiolates, Pro-1 of the β-subunit of CaaD was identified as a general acid catalyst instead of a general base catalyst, thus differentiating it from the rest of the superfamily. 4-OT and YwhB also catalyze dehalogenation, but at a low level. These results suggest that one or both enzymes may be the ancestor(s) of CaaD. Both enzymes are also inhibited by 3-halopropiolates, but by a different mechanism than that observed for CaaD. 2-Hydroxymuconate semialdehyde dehydrogenase (2-HMSD) immediately precedes 4-OT in the meta-fission pathway and is a member of the aldehyde dehydrogenase superfamily. Its catalytic mechanism may utilize a cysteine to form a thiohemiacetal intermediate, which is followed by hydride transfer to NAD+. 2- HMSD has been characterized with several substrates, and site-directed mutagenesis has identified the essential catalytic cysteine. Additionally, 2-HMSD is reversibly inhibited by a product analog. This work sets the stage for further investigaton of its structure and mechanism.