Crystal structure of PqqC from <i>Klebsiella pneumoniae</i> at 2.1 Å resolution

Schwarzenbacher, Robert; Stenner, Frank; Liewen, Heike; Reed, John C.; Liddington, Robert

doi:10.1002/prot.20085

Cited by 18 publications

(25 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3 and 4). Analysis of the PqqC structure shows that the seven ␣-helices provide the scaffold for an active site cavity (19). The cavity is lined with 42 mostly hydrophilic and aromatic residues that are highly conserved within PqqC proteins from different bacteria (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…PqqC from K. pneumoniae (NCBI accession no. X58778) was expressed in E. coli and purified as described elsewhere (19).…”

Section: Methodsmentioning

confidence: 99%

“…The PqqC structure was determined with a selenium-MAD experiment as described (19). Native and complex datasets were collected at beamline 9.1 of the Stanford Synchrotron Radiation Laboratory and processed with the HKL suite (26).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Quinone biogenesis: Structure and mechanism of PqqC, the final catalyst in the production of pyrroloquinoline quinone

Magnússon

Toyama

Saeki

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

113

View full text Add to dashboard Cite

The biosynthesis of pyrroloquinoline quinone (PQQ), a vitamin and redox cofactor of quinoprotein dehydrogenases, is facilitated by an unknown pathway that requires the expression of six genes, pqqA to -F. PqqC, the protein encoded by pqqC, catalyzes the final step in the pathway in a reaction that involves ring cyclization and eight-electron oxidation of 3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicarboxylic-acid to PQQ. Herein, we describe the crystal structures of PqqC and its complex with PQQ and determine the stoichiometry of H2O2 formation and O2 uptake during the reaction. The PqqC structure(s) reveals a compact seven-helix bundle that provides the scaffold for a positively charged active site cavity. Product binding induces a large conformational change, which results in the active site recruitment of amino acid side chains proposed to play key roles in the catalytic mechanism. PqqC is unusual in that it transfers redox equivalents to molecular oxygen without the assistance of a redox active metal or cofactor. The structure of the enzyme-product complex shows additional electron density next to R179 and C5 of PQQ, which can be modeled as O2 or H2O2, indicating a site for oxygen binding. We propose a reaction sequence that involves base-catalyzed cyclization and a series of quinone-quinol tautomerizations that are followed by cycles of O2͞H2O2-mediated oxidations. P yrroloquinoline quinone [4,5-dihydro-4,5-dioxo-1H-pyrrolo-[2,3-f]quinoline-2,7,9-tricarboxylic acid; PQQ ( Fig. 1)] is an aromatic, tricyclic ortho-quinone that serves as the redox cofactor for several bacterial dehydrogenases. Among the best known examples are methanol dehydrogenase and glucose dehydrogenase (1, 2). PQQ belongs to the family of quinone cofactors that has been recognized as the third class of redox cofactors following pyridine nucleotide-and flavin-dependent cofactors (3). Although plants and animals do not produce PQQ themselves, PQQ has invoked considerable interest because of its presence in human milk and its remarkable antioxidant properties (4-6). Recently, the first potential eukaryotic PQQdependent enzyme [aminoadipic 6-semialdehyde-dehydrogenase (AASDH; U26)] has been identified, indicating that PQQ may function as a vitamin in mammals as well (7).Quinone cofactors are generally covalently linked to the polypeptide chain and derived posttranslationally from precursor amino acid residues encoded within their parental polypeptide chain. For example, in copper amine oxidases, topaquinone is formed by a ''self-processing'' oxidation of a specific Tyr residue in the presence of copper ion and molecular oxygen (8,9). PQQ is distinct from the other quinone cofactors in that its biogenesis is independent of its site of action. PQQ is constructed from the amino acids glutamate and tyrosine, as shown in Fig. 1 (10, 11). The PQQ biosynthesis pathway in Klebsiella pneumoniae requires the expression of six genes, designated pqqABCDEF (12). PqqA encodes a 23-residue peptide with conserved glutamate and...

show abstract

Section: Resultsmentioning

confidence: 99%

“…PqqC from K. pneumoniae (NCBI accession no. X58778) was expressed in E. coli and purified as described elsewhere (19).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Quinone biogenesis: Structure and mechanism of PqqC, the final catalyst in the production of pyrroloquinoline quinone

Magnússon

Toyama

Saeki

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

113

View full text Add to dashboard Cite

show abstract

“…1A) was identified more than 25 years ago (4,30), and more recent bioinformatics studies demonstrated a conservation of gene order that includes an occasional gene fusion between PqqD with either the C terminus of PqqC or the N terminus of PqqE (2). However, the description of function for each gene product from this operon has lagged considerably, with PqqC representing the only enzyme within the pathway successfully characterized with regard to structural, spectroscopic, and kinetic properties (5,6,31). Although PqqE had been shown to be a radical SAM enzyme (22), it had not been possible to demonstrate a reaction between the peptide substrate PqqA and PqqE, suggesting either that PqqA must be modified before it can interact with PqqE or that key components of the PqqE system were missing (7).…”

Section: Discussionmentioning

confidence: 99%

Demonstration That the Radical S-Adenosylmethionine (SAM) Enzyme PqqE Catalyzes de Novo Carbon-Carbon Cross-linking within a Peptide Substrate PqqA in the Presence of the Peptide Chaperone PqqD

Barr

Latham

Iavarone

et al. 2016

Journal of Biological Chemistry

108

181

View full text Add to dashboard Cite

The radical S-adenosylmethionine (SAM) protein PqqE is predicted to function in the pyrroloquinoline quinone (PQQ) biosynthetic pathway via catalysis of carbon-carbon bond formation between a glutamate and tyrosine side chain within the small peptide substrate PqqA. We report here that PqqE activity is dependent on the accessory protein PqqD, which was recently shown to bind PqqA tightly. In addition, PqqE activity in vitro requires the presence of a flavodoxin-and flavodoxin reductasebased reduction system, with other reductants leading to an uncoupled cleavage of the co-substrate SAM. These results indicate that PqqE, in conjunction with PqqD, carries out the first step in PQQ biosynthesis: a radical-mediated formation of a new carbon-carbon bond between two amino acid side chains on PqqA.Pyrroloquinoline quinone (PQQ) 2 is employed by a wide variety of bacteria, where it functions as a redox cofactor in substrate oxidation via an alternate (non-glycolytic) pathway for production of cellular ATP (1). Hundreds of bacterial species are thought to produce PQQ, based on the presence in their genomes of the strongly conserved pqq operon (2) (Fig. 1A). Although the existence of this important redox cofactor has been known for decades, knowledge of the biosynthetic pathway for PQQ has remained scant (3, 4). The PQQ molecule derives from the evolutionarily conserved glutamate and tyrosine side chains within a ribosomally produced peptide substrate PqqA (Fig. 1, B and C). To produce PQQ, a large number of chemical modifications are required: (i) the formation of a new carbon-carbon bond between the ␥-carbon of glutamate and the 3-position of tyrosine (labeled in green in Fig. 1C); (ii) the addition of two additional hydroxyl groups to the tyrosine ring; (iii) a condensation between the 4-OH of tyrosine and the backbone amine of glutamate to produce a heterocyclic ring; and (iv) an 8-electron oxidation catalyzed by PqqC, a cofactorless enzyme that converts 3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicarboxylic acid (AHQQ) to PQQ in the final step of the pathway (5-7).The radical SAM enzyme PqqE has been the primary candidate as the catalyst for the formation of the new carbon-carbon bond between glutamate and tyrosine. Radical SAM proteins use the reductive cleavage of S-adenosyl methionine to initiate free radical chemistry, and can accomplish a wide variety of reactions, including carbon-carbon bond formation (8, 9). PqqE is a founding member of the SPASM domain-containing radical SAM proteins, which contain one or more auxiliary clusters in their C-terminal regions, and of which several are known to modify small peptides or proteins (10, 11). Recently, a small (ϳ10 kDa) protein, PqqD, has been shown to form a strong, sub-micromolar K D complex with the peptide substrate PqqA. This complex was further demonstrated to associate with PqqE (12). These findings suggested that PqqD would serve as a chaperone to deliver PqqA to PqqE, functioning as a necessary and heretofore missing componen...

show abstract

“…Although the structures of PqqB, PqqC, and PqqD, which are involved in PQQ biosynthesis, have been solved, the initial steps of the PQQ biosynthesis pathway remain unknown (30,37,38). However, the pqqF gene of K. pneumoniae has been shown to be essential for PQQ biosynthesis in E. coli and E. coli pts (phosphotransferase system) mutants on minimum medium (39).…”

mentioning

confidence: 99%

Crystal Structure and Function of PqqF Protein in the Pyrroloquinoline Quinone Biosynthetic Pathway

Wei

Ran

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

Pyrroloquinoline quinone (PQQ) has received considerable attention due to its numerous important physiological functions. PqqA is a precursor peptide of PQQ with two conserved residues: glutamate and tyrosine. After linkage of the C␥ of glutamate and C⑀ of tyrosine by PqqE, these two residues are hypothesized to be cleaved from PqqA by PqqF. The linked glutamate and tyrosine residues are then used to synthesize PQQ. Here, we demonstrated that the pqqF gene is essential for PQQ biosynthesis as deletion of it eliminated the inhibition of prodigiosin production by glucose. We further determined the crystal structure of PqqF, which has a closed clamshell-like shape. The PqqF consists of two halves composed of an N-and a C-terminal lobe. The PqqF-N and PqqF-C lobes form a chamber with the volume of the cavity of ϳ9400 Å 3 . The PqqF structure conforms to the general structure of inverzincins. Compared with the most thoroughly characterized inverzincin insulin-degrading enzyme, the size of PqqF chamber is markedly smaller, which may define the specificity for its substrate PqqA. Furthermore, the 14-amino acid-residue-long tag formed by the N-terminal tag from expression vector precisely protrudes into the counterpart active site; this N-terminal tag occupies the active site and stabilizes the closed, inactive conformation. His-48, His-52, Glu-129 and His-14 from the N-terminal tag coordinate with the zinc ion. Glu-51 acts as a base catalyst. The observed histidine residue-mediated inhibition may be applicable for the design of a peptide for the inhibition of M16 metalloproteases.Pyrroloquinoline quinone (PQQ), 4 an aromatic orthoquinone, has been recognized as the third class of redox cofactors in addition to the well known cofactors, nicotinamide (NAD(P) ϩ ) and flavin (FAD, FMN) (1). PQQ was first identified from methanol dehydrogenase in methylotrophic bacteria (2), and several bacterial dehydrogenases, such as glucose dehydrogenases, quinate dehydrogenase, and alcohol dehydrogenase, were later found to be quinoproteins (3-5). Recently, sugar oxidoreductase in the basidiomycete Coprinopsis cinerea (6), 2-keto-D-glucose dehydrogenase from Pseudomonas aureofaciens (7) and pyranose dehydrogenase from C. cinerea (8) were all characterized as novel PQQ-dependent enzymes. Free PQQ has been identified in a wide variety of foods (9) and milk (10). PQQ is an essential nutrient for proper growth and development in mice (11). The strong-antioxidant capacity of PQQ protects living cells and biomolecules from oxidative stress in vivo and in vitro (12, 13). Furthermore, PQQ exerts a protective effect against ultraviolet irradiation-induced human dermal fibroblast senescence in vitro (14) and suppresses the serum low density cholesterol level to prevent various diseases (15). In addition, PQQ may improve skin conditions and slow the progression of osteoarthritis (16).The chemical structure of PQQ was determined in 1980 (17). Since then gene clusters involved in the synthesis of PQQ from different bacteria that range from 4 genes ...

show abstract

Crystal structure of PqqC from Klebsiella pneumoniae at 2.1 Å resolution

Cited by 18 publications

References 18 publications

Quinone biogenesis: Structure and mechanism of PqqC, the final catalyst in the production of pyrroloquinoline quinone

Quinone biogenesis: Structure and mechanism of PqqC, the final catalyst in the production of pyrroloquinoline quinone

Demonstration That the Radical S-Adenosylmethionine (SAM) Enzyme PqqE Catalyzes de Novo Carbon-Carbon Cross-linking within a Peptide Substrate PqqA in the Presence of the Peptide Chaperone PqqD

Crystal Structure and Function of PqqF Protein in the Pyrroloquinoline Quinone Biosynthetic Pathway

Contact Info

Product

Resources

About