Crystal Structure of d-Erythronate-4-phosphate Dehydrogenase Complexed with NAD

Ha, Jun Yong; Lee, Ji Hyun; Kim, Kyoung Hoon; Kim, Dojin; Lee, Hyung Ho; Kim, Hye-Kyung; Yoon, Hye‐Jin; Suh, Se Won

doi:10.1016/j.jmb.2006.12.038

Cited by 17 publications

(18 citation statements)

References 33 publications

(40 reference statements)

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“…The protein migrated as an apparent dimer by gel filtration chromatography, in agreement with the dimeric form observed in the crystal structure of the PdxB homolog from Pseudomonas aeruginosa (7). As purified, the enzyme exhibits a prominent spectral signal at 322 nm (Figure 2), suggestive of a nicotino-protein in the reduced state wherein the typical 340 nm absorbance of NADH is significantly blue-shifted in the apolar environment of the protein (8).…”

Section: Resultssupporting

confidence: 81%

“…The tightly bound NAD(P)(H) cofactor in nicotino-enzymes often exists in a highly buried protein environment (25). The crystal structure of PdxB from Pseudomonas aeruginosa (7) (45% identity with PdxB from E. coli ) yields some insight into the nature of the tight binding interaction between PdxB and NAD. NAD binds in an extended conformation in the nucleotide binding domain using numerous specific interactions with PdxB and is completely shielded from the surface.…”

Section: Discussionmentioning

confidence: 99%

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Multiple Turnovers of the Nicotino-Enzyme PdxB Require α-Keto Acids as Cosubstrates

2010

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PdxB catalyzes the second step in the biosynthesis of pyridoxal phosphate by oxidizing 4-phospho-D-erythronate (4PE) to 2-oxo-3-hydroxy-4-phospho-butanoate (OHPB) with concomitant reduction of NAD+ to NADH. PdxB is a nicotino-enzyme wherein the NAD(H) cofactor remains tightly bound to PdxB. It has been a mystery how PdxB performs multiple turnovers since addition of free NAD+ does not re-oxidize the enzyme-bound NADH following conversion of 4PE to OHPB. We have solved this mystery by demonstrating that a variety of physiologically available α-ketoacids serve as oxidants of PdxB to sustain multiple turnovers. In a coupled assay using the next two enzymes of the biosynthetic pathway for pyridoxal phosphate (SerC and PdxA), we have found that α-ketoglutarate, oxaloacetic acid, and pyruvate are equally good substrates for PdxB (kcat/Km values ~ 1 × 104 M-1s-1). The kinetic parameters for the substrate 4PE include a kcat of 1.4 s-1, a Km of 2.9 μM, and a kcat/Km of 6.7 × 106 M-1s-1. Additionally, we have characterized the stereochemistry of α-ketoglutarate reduction by showing that D-2-HGA, but not L-2-HGA, is a competitive inhibitor vs. 4PE and a noncompetitive inhibitor vs. α-ketoglutarate.

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Section: Resultssupporting

confidence: 81%

Section: Discussionmentioning

confidence: 99%

Multiple Turnovers of the Nicotino-Enzyme PdxB Require α-Keto Acids as Cosubstrates

2010

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“…These four residues (Arg251, Ser253, His255, and Lys263) seem to form and ideal phosphate binding pocket (Figure 6) and it is tempting to speculate that these residues interact in a similar manner with the phosphate moiety of the sugar phosphate that donates the carbon to the isocyano product. The appearance of a phosphate from the crystallization solution bound in the physiologically relevant phosphate binding site of a protein in an active site is not unusual27,28,29 and can be used as a means to propose the location of active sites within proteins30. Adjacent to this phosphate binding site is a solvent filled cavity that is also surrounded by well-conserved residues from several of the remaining motifs.…”

Section: Resultsmentioning

confidence: 99%

Three-dimensional Structures of Pseudomonas aeruginosa PvcA and PvcB, Two Proteins Involved in the Synthesis of 2-Isocyano-6,7-dihydroxycoumarin

Drake

Gulick

2008

Journal of Molecular Biology

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Summary The pvcABCD operon of Pseudomonas aeruginosa encodes four proteins (PA2254, PA2255, PA2256, and PA2257) that form a cluster that is responsible for the synthesis of a cyclized isocyano derivative of tyrosine. These proteins, which were originally identified as being responsible for a step in the maturation of the chromophore of the peptide siderophore pyoverdine, have recently been identified as belonging to a family of proteins that produce small organic isonitriles. We report that strains harboring a disruption in the pvcA or pvcB genes are able to grow in iron depleted conditions and to produce pyoverdine. Additionally, we have determined the three dimensional crystal structures of PvcA and PvcB. The structure of PvcA demonstrates a novel enzyme architecture that is built upon a Rossmann fold. We have analyzed the sequence conservation of enzymes within this family and identified six conserved motifs. These regions of the protein cluster around a putative active site cavity. The structure of the PvcB protein confirms it is a member of the Fe2+/α-ketoglutarate dependent oxygenase family of enzymes. The active site of PvcB is compared to the structures of other family members and suggests that a conformational change to order several loops will accompany the binding of ligands.

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“…1) D-lactate, [2][3][4] D-hydroxyisocaproate, 5) D-glycerate, 6) and D-erythronate-4-phosphate 7) dehydrogenases, vancomycin-resistant protein H (VanH), 8,9) glyoxylate reductase (GR), 10) and transcriptional co-repressor CtBP, 11) share a common protein structure and constitute the D-2-HydDH family, 12) together with some non-D-2-HydDHs, such as the formate, 13) phosphite, 14,15) and L-alanine 16) dehydrogenases. It has been reported that only minor structural changes, such as amino acid replacements, cause drastic change, in the enzyme function in this family, as for example, conversions from a D-lactate dehydrogenase (D-LDH) to a D-hydroxyisocaproate dehydrogenase (D-HicDH), 17,18) and even from a formate dehydrogenase to a D-2-HydDH.…”

mentioning

confidence: 99%

A New Family ofD-2-Hydroxyacid Dehydrogenases That ComprisesD-Mandelate Dehydrogenases and 2-Ketopantoate Reductases

Wada

Iwai

Tamura

et al. 2008

Bioscience, Biotechnology, and Biochemistry

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Crystal Structure of d-Erythronate-4-phosphate Dehydrogenase Complexed with NAD

Cited by 17 publications

References 33 publications

Multiple Turnovers of the Nicotino-Enzyme PdxB Require α-Keto Acids as Cosubstrates

Multiple Turnovers of the Nicotino-Enzyme PdxB Require α-Keto Acids as Cosubstrates

Three-dimensional Structures of Pseudomonas aeruginosa PvcA and PvcB, Two Proteins Involved in the Synthesis of 2-Isocyano-6,7-dihydroxycoumarin

A New Family ofD-2-Hydroxyacid Dehydrogenases That ComprisesD-Mandelate Dehydrogenases and 2-Ketopantoate Reductases

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