Cloning, purification and crystallization of<i>Thermus thermophilus</i>proline dehydrogenase

White, Tommi; Tanner, John J.

doi:10.1107/s1744309105019779

Cited by 5 publications

(13 citation statements)

References 30 publications

(32 reference statements)

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“…PRODH Kinetic Characterization-TtPRODH was expressed in Escherichia coli and purified as described previously (20). The purified enzyme was dialyzed into buffer containing 50 mM Tris-HCl, 50 mM NaCl, 0.5 mM EDTA, 0.5 mM DTT, and 5% glycerol at pH 8.0 and stored at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…Se-Met TtPRODH was crystallized using procedures described previously for native TtPRODH (20). Briefly, crystals were grown at room temperature in sitting drops by mixing equal volumes of the enzyme (2 l) and reservoir (2 l) solutions.…”

Section: Expression Purification and Crystallization Of Se-metmentioning

confidence: 99%

“…The detergent extraction step was repeated once or twice until no additional TtPRODH was liberated. The extracted protein was then purified using nickel-nitrilotriacetic acid chromatography as described previously (20).…”

Section: Expression Purification and Crystallization Of Se-metmentioning

confidence: 99%

“…Se-Met TtPRODH was purified using procedures described for purification of TtPRODH (20), except for the following modifications. As observed for native TtPRODH, the pellet obtained after centrifugation of lysed cells was bright yellow, indicating that a significant amount of Se-Met TtPRODH was bound to the cell debris in addition to Se-Met TtPRODH in the supernatant.…”

Section: Expression Purification and Crystallization Of Se-metmentioning

confidence: 99%

“…Moreover, the bacterial enzymes are convenient systems for studying protein-protein interactions and intermolecular channeling. To begin exploring these questions, we previously reported the cloning, isolation and crystallization of the monofunctional PRODH from Thermus thermophilus (TtPRODH) (20). Here we report the crystal structure and kinetic characterization of this enzyme.…”

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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: Methodsmentioning

confidence: 99%

Section: Expression Purification and Crystallization Of Se-metmentioning

confidence: 99%

Section: Expression Purification and Crystallization Of Se-metmentioning

confidence: 99%

Section: Expression Purification and Crystallization Of Se-metmentioning

confidence: 99%

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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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High yields of active Thermus thermophilus proline dehydrogenase are obtained using maltose‐binding protein as a solubility tag

Huijbers

Berkel

2015

Biotechnology Journal

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Proline dehydrogenase (ProDH) catalyzes the FAD-dependent oxidation of proline to Δ(1) -pyrroline-5-carboxylate, the first step of proline catabolism in many organisms. Next to being involved in a number of physiological processes, ProDH is of interest for practical applications because the proline imino acid can serve as a building block for a wide range of peptides and antibiotics. ProDH is a membrane-associated protein and recombinant soluble forms of the enzyme have only been obtained in limited amounts. We here report on the heterologous production of ProDH from Thermus thermophilus (TtProDH) in Escherichia coli. Using maltose-binding protein as solubility tag, high yields of active holoenzyme are obtained. Native TtProDH can be produced from cleaving the purified fusion protein with trypsin. Size-exclusion chromatography shows that fused and clipped TtProDH form oligomers. Thermal stability and co-solvent tolerance indicate the conformational robustness of TtProDH. These properties together with the high yield make TtProDH attractive for industrial applications.

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Structural Basis for the Inactivation of Thermus thermophilus Proline Dehydrogenase by N-Propargylglycine^,

et al. 2008

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The flavoenzyme proline dehydrogenase catalyzes the first step of proline catabolism, the oxidation of proline to pyrroline-5-carboxylate. Here we report the first crystal structure of an irreversibly inactivated proline dehydrogenase. The 1.9 A resolution structure of Thermus thermophilus proline dehydrogenase inactivated by the mechanism-based inhibitor N-propargylglycine shows that N5 of the flavin cofactor is covalently connected to the -amino group of Lys99 via a three-carbon linkage, consistent with the mass spectral analysis of the inactivated enzyme. The isoalloxazine ring has a butterfly angle of 25 degrees , which suggests that the flavin cofactor is reduced. Two mechanisms can account for these observations. In both, N-propargylglycine is oxidized to N-propargyliminoglycine. In one mechanism, this alpha,beta-unsaturated iminium compound is attacked by the N5 atom of the now reduced flavin to produce a 1,4-addition product. Schiff base formation between Lys99 and the imine of the 1,4-addition product releases glycine and links the enzyme to the modified flavin. In the second mechanism, hydrolysis of N-propargyliminoglycine yields propynal and glycine. A 1,4-addition reaction with propynal coupled with Schiff base formation between Lys99 and the carbonyl group tethers the enzyme to the flavin via a three-carbon chain. The presumed nonenzymatic hydrolysis of N-propargyliminoglycine and the subsequent rebinding of propynal to the enzyme make the latter mechanism less likely.

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Cloning, purification and crystallization ofThermus thermophilusproline dehydrogenase

Cited by 5 publications

References 30 publications

Structure and Kinetics of Monofunctional Proline Dehydrogenase from Thermus thermophilus

Structure and Kinetics of Monofunctional Proline Dehydrogenase from Thermus thermophilus

High yields of active Thermus thermophilus proline dehydrogenase are obtained using maltose‐binding protein as a solubility tag

Structural Basis for the Inactivation of Thermus thermophilus Proline Dehydrogenase by N-Propargylglycine^,

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Cloning, purification and crystallization ofThermus thermophilusproline dehydrogenase

Cited by 5 publications

References 30 publications

Structure and Kinetics of Monofunctional Proline Dehydrogenase from Thermus thermophilus

Structure and Kinetics of Monofunctional Proline Dehydrogenase from Thermus thermophilus

High yields of active Thermus thermophilus proline dehydrogenase are obtained using maltose‐binding protein as a solubility tag

Structural Basis for the Inactivation of Thermus thermophilus Proline Dehydrogenase by N-Propargylglycine,

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Structural Basis for the Inactivation of Thermus thermophilus Proline Dehydrogenase by N-Propargylglycine^,