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 journalArticlepeer-review

50 Scopus citations


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
Issue number2
StatePublished - Jan 16 2007

ASJC Scopus subject areas

  • Biochemistry


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