Redox-Induced Changes in Flavin Structure and Roles of Flavin N(5) and the Ribityl 2‘-OH Group in Regulating PutA−Membrane Binding<sup>,</sup>

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

doi:10.1021/bi061935g

Cited by 51 publications

(141 citation statements)

References 43 publications

(84 reference statements)

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“…In the oxidized state, it remains in the cytoplasm and represses transcription of the putA and putP (encodes a proline transporter) genes (15,46,47). We recently showed that reduction of FAD in PutA86 -669 triggers rupture of the hydrogen bond between the ribityl 2Ј-OH and Arg 556 , causing rotation of the 2Ј-OH so that it is tucked below, and hydrogen bonded to, the FAD N1 (39). We further showed that the 2Ј-OH-Arg 556 hydrogen bond is a structural constraint that prevents oxidized PutA from binding the membrane (39).…”

Section: Discussionmentioning

confidence: 99%

“…We recently showed that reduction of FAD in PutA86 -669 triggers rupture of the hydrogen bond between the ribityl 2Ј-OH and Arg 556 , causing rotation of the 2Ј-OH so that it is tucked below, and hydrogen bonded to, the FAD N1 (39). We further showed that the 2Ј-OH-Arg 556 hydrogen bond is a structural constraint that prevents oxidized PutA from binding the membrane (39). Interestingly, we find here that the 2Ј-OH of oxidized TtPRODH is tucked below the FAD N1, that is, cocked in the membrane-binding position (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…We recently reported the structure of dithionite-reduced PutA86 -669 and showed that reduction of the FAD induces a 22°b end of the isoalloxazine and rotation of the 2Ј-OH by 90°so that it is tucked under the pyrimidine ring and forms a hydrogen bond to the FAD N1 (39). Thus, the conformations of the 2Ј-OH groups of oxidized TtPRODH and reduced PutA86 -669 are identical (Fig.…”

Section: Aspmentioning

confidence: 92%

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Structure and Kinetics of Monofunctional Proline Dehydrogenase from Thermus thermophilus

White¹,

Krishnan

Becker

et al. 2007

Journal of Biological Chemistry

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145

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Proline dehydrogenase (PRODH) and ⌬ 1 -pyrroline-5-carboxylate dehydrogenase (P5CDH) catalyze the two-step oxidation of proline to glutamate. They are distinct monofunctional enzymes in all eukaryotes and some bacteria but are fused into bifunctional enzymes known as proline utilization A (PutA) in other bacteria. Here we report the first structure and biochemical data for a monofunctional PRODH. The 2.0-Å resolution structure of Thermus thermophilus PRODH reveals a distorted (␤␣) 8 barrel catalytic core domain and a hydrophobic ␣-helical domain located above the carboxylterminal ends of the strands of the barrel. Although the catalytic core is similar to that of the PutA PRODH domain, the FAD conformation of T. thermophilus PRODH is remarkably different and likely reflects unique requirements for membrane association and communication with P5CDH. Also, the FAD of T. thermophilus PRODH is highly solvent-exposed compared with PutA due to a 4-Å shift of helix 8. Structurebased sequence analysis of the PutA/PRODH family led us to identify nine conserved motifs involved in cofactor and substrate recognition. Biochemical studies show that the midpoint potential of the FAD is ؊75 mV and the kinetic parameters for proline are K m ‫؍‬ 27 mM and k cat ‫؍‬ 13 s ؊1 .3,4-Dehydro-L-proline was found to be an efficient substrate, and L-tetrahydro-2-furoic acid is a competitive inhibitor (K I ‫؍‬ 1.0 mM). Finally, we demonstrate that T. thermophilus PRODH reacts with O 2 producing superoxide. This is significant because superoxide production underlies the role of human PRODH in p53-mediated apoptosis, implying commonalities between eukaryotic and bacterial monofunctional PRODHs.Oxidation of amino acids is a central part of energy metabolism. The oxidative pathway for proline consists of two enzymatic steps and an intervening nonenzymatic equilibrium (Scheme 1) (1, 2). The first enzymatic step transforms proline to ⌬ 1 -pyrroline-5-carboxylate (P5C), 2 which is non-enzymatically hydrolyzed to glutamic semialdehyde. The semialdehyde is oxidized in the second enzymatic step to glutamate.This 4-electron transformation of proline is common to all organisms, but the enzymes of proline catabolism differ widely among the three kingdoms of life. Amino acid sequence analysis shows that bacteria and eukaryotes share a common set of proline catabolic enzymes called proline dehydrogenase (PRODH) and P5C dehydrogenase (P5CDH). Studies of the bacterial enzymes have shown that PRODH is an FAD-dependent enzyme with a (␤␣) 8 barrel catalytic core (3, 4), and P5CDH is an NAD ϩ -dependent Rossmann fold enzyme featuring a nucleophilic Cys (5). These enzymes are unrelated in sequence and structure to hyperthermophilic archaeal proline catabolic enzymes, which appear in unique hetero-tetrameric and hetero-octameric complexes (6).An intriguing aspect of proline catabolism in eukaryotes and bacteria is that PRODH and P5CDH are separate enzymes in some organisms, whereas the two enzymes are fused in other organisms. The traditional view has been that P...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Aspmentioning

confidence: 92%

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Structure and Kinetics of Monofunctional Proline Dehydrogenase from Thermus thermophilus

White¹,

Krishnan

Becker

et al. 2007

Journal of Biological Chemistry

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145

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“…The flavoprotein PutA acts as a transcriptional repressor of the proline utilization (put) operon by binding to an operator when intracellular proline concentrations are low (48 -50). However, high intracellular proline concentrations have been shown to alter the function of PutA driven by the reduction of PutA-bound FAD (51)(52)(53). Reduced PutA associates with the membrane and catalyzes the two-step oxidation of proline to glutamate.…”

Section: Demonstration Of a Direct Electron Transfer From Reduced Menmentioning

confidence: 99%

Biochemical Studies of Klebsiella pneumoniae NifL Reduction Using Reconstituted Partial Anaerobic Respiratory Chains of Wolinella succinogenes

Thummer

Klimmek

Schmitz

2007

Journal of Biological Chemistry

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In the diazotroph Klebsiella pneumoniae the flavoprotein NifL inhibits the activity of the nif-specific transcriptional activator NifA in response to molecular oxygen and combined nitrogen. Sequestration of reduced NifL to the cytoplasmic membrane under anaerobic and nitrogen-limited conditions impairs inhibition of cytoplasmic NifA by NifL. To analyze whether NifL is reduced by electrons directly derived from the reduced menaquinone pool, we studied NifL reduction using artificial membrane systems containing purified components of the anaerobic respiratory chain of Wolinella succinogenes. In this in vitro assay using proteoliposomes containing purified formate dehydrogenase and purified menaquinone (MK 6 ) or 8-methylmenaquinone (MMK 6 ) from W. succinogenes, reduction of purified NifL was achieved by formate oxidation. Furthermore, the respective reduction rates, which were determined using equal amounts of NifL, have been shown to be directly dependent on the concentration of both formate dehydrogenase and menaquinones incorporated into the proteoliposomes, demonstrating a direct electron transfer from menaquinone to NifL. When purified hydrogenase and MK 6 from W. succinogenes were inserted into the proteoliposomes, NifL was reduced with nearly the same rate by hydrogen oxidation. In both cases reduced NifL was found to be highly associated to the proteoliposomes, which is in accordance with our previous findings in vivo. On the bases of these experiments, we propose that the redox state of the menaquinone pool is the redox signal for nif regulation in K. pneumoniae by directly transferring electrons onto NifL under anaerobic conditions.

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“…These proteins were chosen because recent biochemical and structural studies of these flavin switch proteins have provided novel details into how cofactor reduction=modification leads to the propagation of conformational and functional changes (50,69,83,90). Two of the proteins, VVD and NifL, contain a flavin-binding Per Arnt Sim (PAS) domain (64,66).…”

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confidence: 99%

Flavin Redox Switching of Protein Functions

Becker

Zhu

Moxley

2011

Antioxidants & Redox Signaling

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Flavin cofactors impart remarkable catalytic diversity to enzymes, enabling them to participate in a broad array of biological processes. The properties of flavins also provide proteins with a versatile redox sensor that can be utilized for converting physiological signals such as cellular metabolism, light, and redox status into a unique functional output. The control of protein functions by the flavin redox state is important for transcriptional regulation, cell signaling pathways, and environmental adaptation. A significant number of proteins that have flavin redox switches are found in the Per-Arnt-Sim (PAS) domain family and include flavoproteins that act as photosensors and respond to changes in cellular redox conditions. Biochemical and structural studies of PAS domain flavoproteins have revealed key insights into how flavin redox changes are propagated to the surface of the protein and translated into a new functional output such as the binding of a target protein in a signaling pathway. Mechanistic details of proteins unrelated to the PAS domain are also emerging and provide novel examples of how the flavin redox state governs protein-membrane interactions in response to appropriate stimuli. Analysis of different flavin switch proteins reveals shared mechanistic themes for the regulation of protein structure and function by flavins.

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Redox-Induced Changes in Flavin Structure and Roles of Flavin N(5) and the Ribityl 2‘-OH Group in Regulating PutA−Membrane Binding^,

Cited by 51 publications

References 43 publications

Structure and Kinetics of Monofunctional Proline Dehydrogenase from Thermus thermophilus

Structure and Kinetics of Monofunctional Proline Dehydrogenase from Thermus thermophilus

Biochemical Studies of Klebsiella pneumoniae NifL Reduction Using Reconstituted Partial Anaerobic Respiratory Chains of Wolinella succinogenes

Flavin Redox Switching of Protein Functions

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Redox-Induced Changes in Flavin Structure and Roles of Flavin N(5) and the Ribityl 2‘-OH Group in Regulating PutA−Membrane Binding,

Cited by 51 publications

References 43 publications

Structure and Kinetics of Monofunctional Proline Dehydrogenase from Thermus thermophilus

Structure and Kinetics of Monofunctional Proline Dehydrogenase from Thermus thermophilus

Biochemical Studies of Klebsiella pneumoniae NifL Reduction Using Reconstituted Partial Anaerobic Respiratory Chains of Wolinella succinogenes

Flavin Redox Switching of Protein Functions

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Redox-Induced Changes in Flavin Structure and Roles of Flavin N(5) and the Ribityl 2‘-OH Group in Regulating PutA−Membrane Binding^,