Document Detail


Crystal structures and kinetics of monofunctional proline dehydrogenase provide insight into substrate recognition and conformational changes associated with flavin reduction and product release.
MedLine Citation:
PMID:  23151026     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Proline dehydrogenase (PRODH) catalyzes the FAD-dependent oxidation of proline to Δ(1)-pyrroline-5-carboxylate, which is the first step of proline catabolism. Here, we report the structures of proline dehydrogenase from Deinococcus radiodurans in the oxidized state complexed with the proline analogue L-tetrahydrofuroic acid and in the reduced state with the proline site vacant. The analogue binds against the si face of the FAD isoalloxazine and is protected from bulk solvent by helix α8 and the β1-α1 loop. The FAD ribityl chain adopts two conformations in the E-S complex, which is unprecedented for flavoenzymes. One of the conformations is novel for the PRODH superfamily and may contribute to the low substrate affinity of Deinococcus PRODH. Reduction of the crystalline enzyme-inhibitor complex causes profound structural changes, including 20° butterfly bending of the isoalloxazine, crankshaft rotation of the ribityl, shifting of α8 by 1.7 Å, reconfiguration of the β1-α1 loop, and rupture of the Arg291-Glu64 ion pair. These changes dramatically open the active site to facilitate product release and allow electron acceptors access to the reduced flavin. The structures suggest that the ion pair, which is conserved in the PRODH superfamily, functions as the active site gate. Mutagenesis of Glu64 to Ala decreases the catalytic efficiency 27-fold, which demonstrates the importance of the gate. Mutation of Gly63 decreases the efficiency 140-fold, which suggests that flexibility of the β1-α1 loop is essential for optimal catalysis. The large conformational changes that are required to form the E-S complex suggest that conformational selection plays a role in substrate recognition.
Authors:
Min Luo; Benjamin W Arentson; Dhiraj Srivastava; Donald F Becker; John J Tanner
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2012-12-05
Journal Detail:
Title:  Biochemistry     Volume:  51     ISSN:  1520-4995     ISO Abbreviation:  Biochemistry     Publication Date:  2012 Dec 
Date Detail:
Created Date:  2012-12-18     Completed Date:  2013-02-19     Revised Date:  2014-01-09    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  10099-108     Citation Subset:  IM    
Data Bank Information
Bank Name/Acc. No.:
PDB/4H6Q;  4H6R
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Crystallization
Crystallography, X-Ray
Deinococcus / enzymology
Flavin-Adenine Dinucleotide / metabolism
Flavins / metabolism
Furans / metabolism
Kinetics
Models, Molecular
Molecular Conformation
Oxidation-Reduction
Proline Oxidase / antagonists & inhibitors,  chemistry*,  metabolism*
Grant Support
ID/Acronym/Agency:
GM065546/GM/NIGMS NIH HHS; P41 RR015301/RR/NCRR NIH HHS; R01 GM065546/GM/NIGMS NIH HHS; RR-15301/RR/NCRR NIH HHS
Chemical
Reg. No./Substance:
0/Flavins; 0/Furans; 146-14-5/Flavin-Adenine Dinucleotide; EC 1.5.3.-/Proline Oxidase
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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