Proline utilization A (PutA) from Bradyrhizobium japonicum (BjPutA) is a bifunctional avoenzyme that catalyzes the
oxidation of proline to glutamate using fused proline dehydrogenase (PRODH) and ∆1-pyrroline-5-carboxylate
dehydrogenase (P5CDH) domains. Recent crystal structures and kinetic data suggest an intramolecular channel connects the two active sites,
promoting substrate channeling of the intermediate P5C from the PRODH domain to the P5CDH domain. In this work several mutations were
made along the channel in an effort to block passage of P5C to the second active site. Analysis of several site-specic mutants in the substrate
channel of BjPutA revealed an important role for D779 in the channeling path. BjPutA mutants D779Y and D779W signicantly decreased the
overall PRODH-P5CDH channeling reaction indicating that bulky mutations at residue D779 impede travel of P5C through the channel.
Interestingly, D779Y and D779W also exhibited lower P5CDH activity, suggesting that exogenous P5C must enter the channel upstream of
D779. Replacing D779 with a smaller residue (D779A) had no effect on the catalytic and channeling properties of BjPutA showing that the
carboxylate group of D779 is not essential for channeling. An identical mutation at D778 (D778Y) did not impact BjPutA channeling activity.
Thus, D779 is optimally orientated so that replacement with the larger side chains of Tyr/Trp blocks P5C movment through the channel. The
kinetic data reveal not only that bulky mutations at residue D779 hinder passage of P5C to the second active site, but also P5C must use the
channel to efciently access the P5CDH domain. Moreover, these mutants may be used to learn more about the hydrolysis event that is thought to
take place within the channel
Kinetic characterization of proline utilization A (PutA) using proline analogues
