proline utilization a
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2021 ◽  
Vol 712 ◽  
pp. 109025
Author(s):  
David A. Korasick ◽  
Shelbi L. Christgen ◽  
Insaf A. Qureshi ◽  
Donald F. Becker ◽  
John J. Tanner

2021 ◽  
pp. 49-52
Author(s):  
Anand Shanker Singh ◽  
G. Radhika ◽  
R. Praveen Kumar ◽  
Debarshi Jana

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-specic mutants in the substrate channel of BjPutA revealed an important role for D779 in the channeling path. BjPutA mutants D779Y and D779W signicantly 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 efciently 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


2021 ◽  
Vol 35 (S1) ◽  
Author(s):  
YiZi Mao ◽  
Javier Seravalli ◽  
John Tanner ◽  
Donald Becker

2018 ◽  
Vol 114 (12) ◽  
pp. 2833-2843 ◽  
Author(s):  
David A. Korasick ◽  
Ashley C. Campbell ◽  
Shelbi L. Christgen ◽  
Srinivas Chakravarthy ◽  
Tommi A. White ◽  
...  

Molecules ◽  
2017 ◽  
Vol 23 (1) ◽  
pp. 32 ◽  
Author(s):  
David Korasick ◽  
Travis Pemberton ◽  
Benjamin Arentson ◽  
Donald Becker ◽  
John Tanner

Biochemistry ◽  
2017 ◽  
Vol 56 (47) ◽  
pp. 6292-6303 ◽  
Author(s):  
Shelbi L. Christgen ◽  
Weidong Zhu ◽  
Nikhilesh Sanyal ◽  
Bushra Bibi ◽  
John J. Tanner ◽  
...  

2017 ◽  
Vol 632 ◽  
pp. 142-157 ◽  
Author(s):  
Li-Kai Liu ◽  
Donald F. Becker ◽  
John J. Tanner

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