4Q71: Crystal Structure Of Bradyrhizobium Japonicum Proline Utilization A (puta) Mutant D779w

Citation:
Abstract
Proline utilization A from Bradyrhizobium japonicum (BjPutA) is a bifunctional flavoenzyme that catalyzes the oxidation of proline to glutamate using fused proline dehydrogenase (PRODH) and Delta(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 Delta(1)-pyrroline-5-carboxylate/glutamate-gamma-semialdehyde (P5C/GSA). In this work, the structure of the channel was explored by inserting large side chain residues at four positions along the channel in BjPutA. Kinetic analysis of the different mutants revealed replacement of D779 with Tyr (D779Y) or Trp (D779W) significantly decreased the overall rate of the PRODH-P5CDH channeling reaction. X-ray crystal structures of D779Y and D779W revealed that the large side chains caused a constriction in the central section of the tunnel, thus likely impeding the travel of P5C/GSA in the channel. The D779Y and D779W mutants have PRODH activity similar to that of wild-type BjPutA but exhibit significantly lower P5CDH activity, suggesting that exogenous P5C/GSA enters the channel upstream of Asp779. Replacement of nearby Asp778 with Tyr (D778Y) did not impact BjPutA channeling activity. Consistent with the kinetic results, the X-ray crystal structure of D778Y shows that the main channel pathway is not impacted; however, an off-cavity pathway is closed off from the channel. These findings provide evidence that the off-cavity pathway is not essential for substrate channeling in BjPutA.
PDB ID: 4Q71Download
MMDB ID: 122198
PDB Deposition Date: 2014/4/23
Updated in MMDB: 2014/09
Experimental Method:
x-ray diffraction
Resolution: 2.2  Å
Source Organism:
Similar Structures:
Biological Unit for 4Q71: tetrameric; determined by author and by software (PISA)
Molecular Components in 4Q71
Label Count Molecule
Proteins (4 molecules)
4
Proline Dehydrogenase
Molecule annotation
Chemicals (24 molecules)
1
4
2
12
3
8
* Click molecule labels to explore molecular sequence information.

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