4Q71

Crystal Structure of Bradyrhizobium japonicum Proline Utilization A (PutA) Mutant D779W

4Q71 の概要

• エントリーDOI: 10.2210/pdb4q71/pdb
• 関連するPDBエントリー: 4Q72 4Q73
• 分子名称: Proline dehydrogenase, FLAVIN-ADENINE DINUCLEOTIDE, SULFATE ION, ... (5 entities in total)
• 機能のキーワード: proline utilization a, puta, beta-alpha barrel, rossmann fold, proline dehydrogenase, 1-pyrroline-5-carboxylate dehydrogenase, fad binding, oxidoreductase
• 由来する生物種: Bradyrhizobium diazoefficiens USDA 110
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 218053.79

構造登録者

Tanner, J.J.,Luo, M.,Pemberton, T.A. (登録日: 2014-04-23, 公開日: 2014-08-06, 最終更新日: 2023-09-20)

主引用文献

Arentson, B.W.,Luo, M.,Pemberton, T.A.,Tanner, J.J.,Becker, D.F.
Kinetic and Structural Characterization of Tunnel-Perturbing Mutants in Bradyrhizobium japonicum Proline Utilization A.
Biochemistry, 53:5150-5161, 2014

PubMed 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 Δ(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 Δ(1)-pyrroline-5-carboxylate/glutamate-γ-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.

PubMed: 25046425
DOI: 10.1021/bi5007404

実験手法

X-RAY DIFFRACTION (2.2 Å)