Redox-induced changes in flavin structure and roles of flavin N(5) and the ribityl 2′-OH group in regulating PutA-membrane binding

Weimin Zhang, Min Zhang, Weidong Zhu, Yuzhen Zhou, Srimevan Wanduragala, Dustin Rewinkel, John J. Tanner, Donald F Becker

Research output: Contribution to journalArticle

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Abstract

PutA is a novel flavoprotein in Escherichia coli that switches from a transcriptional repressor to a membrane-bound proline catabolic enzyme. Previous crystallographic studies of the PutA proline dehydrogenase (PRODH) domain under oxidizing conditions revealed that FAD N(5) and the ribityl 2′-OH group form hydrogen bonds with Arg431 and Arg556, respectively. Here we identify molecular interactions in the PutA PRODH active site that underlie redox-dependent functional switching of PutA. We report that reduction of the PRODH domain induces major structural changes in the FAD cofactor, including a 22° bend of the isoalloxazine ring along the N(5)-N(10) axis, crankshaft rotation of the upper part of the ribityl chain, and formation of a new hydrogen bond network involving the ribityl 2′-OH group, FAD N(1), and Gly435. The roles of the FAD 2′-OH group and the FAD N(5)-Arg431 hydrogen bond pair in regulating redox-dependent PutA-membrane associations were tested using FAD analogues and site-directed mutagenesis. Kinetic membrane binding measurements and cell-based reporter gene assays of modified PutA proteins show that disrupting the FAD N(5)-Arg431 interaction impairs the reductive activation of PutA-membrane binding. We also show that the FAD 2′-OH group acts as a redox-sensitive toggle switch that controls PutA-membrane binding. These results illustrate a new versatility of the ribityl chain in flavoprotein mechanisms.

Original languageEnglish (US)
Pages (from-to)483-491
Number of pages9
JournalBiochemistry
Volume46
Issue number2
DOIs
StatePublished - Jan 16 2007

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Flavin-Adenine Dinucleotide
Oxidation-Reduction
Membranes
Proline Oxidase
Flavoproteins
Hydrogen
Hydrogen bonds
Pair Bond
Switches
Mutagenesis
Crankshafts
Molecular interactions
4,6-dinitro-o-cresol
Site-Directed Mutagenesis
Reporter Genes
Proline
Escherichia coli
Assays
Catalytic Domain
Genes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Redox-induced changes in flavin structure and roles of flavin N(5) and the ribityl 2′-OH group in regulating PutA-membrane binding. / Zhang, Weimin; Zhang, Min; Zhu, Weidong; Zhou, Yuzhen; Wanduragala, Srimevan; Rewinkel, Dustin; Tanner, John J.; Becker, Donald F.

In: Biochemistry, Vol. 46, No. 2, 16.01.2007, p. 483-491.

Research output: Contribution to journalArticle

Zhang, Weimin ; Zhang, Min ; Zhu, Weidong ; Zhou, Yuzhen ; Wanduragala, Srimevan ; Rewinkel, Dustin ; Tanner, John J. ; Becker, Donald F. / Redox-induced changes in flavin structure and roles of flavin N(5) and the ribityl 2′-OH group in regulating PutA-membrane binding. In: Biochemistry. 2007 ; Vol. 46, No. 2. pp. 483-491.
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