Probing a hydrogen bond pair and the FAD redox properties in the proline dehydrogenase domain of Escherichia coli PutA

Berevan A. Baban, Madhavan P. Vinod, John J. Tanner, Donald F Becker

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

The PutA flavoprotein from Escherichia coli combines DNA-binding, proline dehydrogenase (PRODH), and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) activities onto a single polypeptide. Recently, an X-ray crystal structure of PutA residues 87-612 was solved which identified a D370-Y540 hydrogen bond pair in the PRODH active site that appears to have an important role in shaping proline binding and the FAD redox environment. To examine the role of D370-Y540 in the PRODH active site, mutants D370A, Y540F, and D370A/Y540F were characterized in a form of PutA containing only residues 86-601 (PutA86-601) designed to mimic the known structural region of PutA (87-612). Disruption of the D370-Y540 pair only slightly diminished k cat, while more noticeable affects were observed in Km. The mutant D370A/Y540F showed the most significant changes in the pH dependence of kcat/Km and Km relative to wild-type PutA86-601 with an apparent pKa value of about 8.2 for the pH-dependent decrease in Km. From the pH profile of D370A/Y540F inhibition by L-tetrahydro-2-furoic acid (L-THFA), the pH dependency of K m in D370A/Y540F is interpreted as resulting from the deprotonation of the proline amine in the E-S complex. Replacement of D370 and Y540 produces divergent effects on the Em for bound FAD. At pH 7.0, Em values of -0.026, -0.089 and -0.042 V were determined for the two-electron reduction of bound FAD in D370A, Y540F and D370A/Y540F, respectively. The 40-mV positive shift in Em determined for D370A relative to wild-type PutA86-601 (Em=-0.066 V, pH 7.0) indicates D370 has a key role in modulating the FAD redox environment.

Original languageEnglish (US)
Pages (from-to)49-59
Number of pages11
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1701
Issue number1-2
DOIs
StatePublished - Sep 1 2004

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Proline Oxidase
Pair Bond
Flavin-Adenine Dinucleotide
Escherichia coli
Oxidation-Reduction
Hydrogen
Hydrogen bonds
Proline
1-Pyrroline-5-Carboxylate Dehydrogenase
Flavoproteins
Deprotonation
Catalytic Domain
Amines
Crystal structure
X rays
Peptides
Electrons
DNA
Cats
X-Rays

Keywords

  • Active site residue
  • FAD
  • FAD redox potential
  • NAD
  • P5CDH
  • PRODH
  • Proline dehydrogenase
  • flavin adenine dinucleotide
  • nicotinamide adenine dinucleotide
  • proline dehydrogenase
  • proline utilization
  • put

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Probing a hydrogen bond pair and the FAD redox properties in the proline dehydrogenase domain of Escherichia coli PutA. / Baban, Berevan A.; Vinod, Madhavan P.; Tanner, John J.; Becker, Donald F.

In: Biochimica et Biophysica Acta - Proteins and Proteomics, Vol. 1701, No. 1-2, 01.09.2004, p. 49-59.

Research output: Contribution to journalArticle

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AU - Vinod, Madhavan P.

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AU - Becker, Donald F

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AB - The PutA flavoprotein from Escherichia coli combines DNA-binding, proline dehydrogenase (PRODH), and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) activities onto a single polypeptide. Recently, an X-ray crystal structure of PutA residues 87-612 was solved which identified a D370-Y540 hydrogen bond pair in the PRODH active site that appears to have an important role in shaping proline binding and the FAD redox environment. To examine the role of D370-Y540 in the PRODH active site, mutants D370A, Y540F, and D370A/Y540F were characterized in a form of PutA containing only residues 86-601 (PutA86-601) designed to mimic the known structural region of PutA (87-612). Disruption of the D370-Y540 pair only slightly diminished k cat, while more noticeable affects were observed in Km. The mutant D370A/Y540F showed the most significant changes in the pH dependence of kcat/Km and Km relative to wild-type PutA86-601 with an apparent pKa value of about 8.2 for the pH-dependent decrease in Km. From the pH profile of D370A/Y540F inhibition by L-tetrahydro-2-furoic acid (L-THFA), the pH dependency of K m in D370A/Y540F is interpreted as resulting from the deprotonation of the proline amine in the E-S complex. Replacement of D370 and Y540 produces divergent effects on the Em for bound FAD. At pH 7.0, Em values of -0.026, -0.089 and -0.042 V were determined for the two-electron reduction of bound FAD in D370A, Y540F and D370A/Y540F, respectively. The 40-mV positive shift in Em determined for D370A relative to wild-type PutA86-601 (Em=-0.066 V, pH 7.0) indicates D370 has a key role in modulating the FAD redox environment.

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