Kinetic and structural characterization of tunnel-perturbing mutants in Bradyrhizobium japonicum proline utilization A

Benjamin W. Arentson, Min Luo, Travis A. Pemberton, John J. Tanner, Donald F Becker

Research output: Contribution to journalArticle

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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 Δ1-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 Δ1-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.

Original languageEnglish (US)
Pages (from-to)5150-5161
Number of pages12
JournalBiochemistry
Volume53
Issue number31
DOIs
StatePublished - Aug 12 2014

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Bradyrhizobium
Proline
Proline Oxidase
Tunnels
Glutamic Acid
Kinetics
Crystal structure
1-Pyrroline-5-Carboxylate Dehydrogenase
Oxidoreductases
X-Rays
X rays
Substrates
Constriction
Catalytic Domain
delta-1-pyrroline-5-carboxylate
Oxidation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kinetic and structural characterization of tunnel-perturbing mutants in Bradyrhizobium japonicum proline utilization A. / Arentson, Benjamin W.; Luo, Min; Pemberton, Travis A.; Tanner, John J.; Becker, Donald F.

In: Biochemistry, Vol. 53, No. 31, 12.08.2014, p. 5150-5161.

Research output: Contribution to journalArticle

Arentson, Benjamin W. ; Luo, Min ; Pemberton, Travis A. ; Tanner, John J. ; Becker, Donald F. / Kinetic and structural characterization of tunnel-perturbing mutants in Bradyrhizobium japonicum proline utilization A. In: Biochemistry. 2014 ; Vol. 53, No. 31. pp. 5150-5161.
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AB - 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 Δ1-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 Δ1-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.

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