Structure of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase from<i>Pseudomonas putida</i>

Bell, Brittnaie J.; Watanabe, Leandra; Rios-Steiner, J.; Tulinsky, A.; Lebioda, Lukasz; Arni, R.K.

doi:10.1107/s0907444903013192

Cited by 8 publications

(13 citation statements)

References 15 publications

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“…KDPGA from H. volcanii showed an unusual oligomeric structure constituting a dodecamer. In contrast, KDPGAs from Z. mobilis, E. coli, and Pseudomonas putida constitute homotrimeric structures of 21-to 24-kDa subunits (41)(42)(43). In accordance with the high sequence similarity of KD-PGA from H. volcanii with that of E. coli, both enzymes show high specificity for KDPG (40).…”

Section: Fig 5 Growth Analysis Of the H Volcanii Gad Deletion Mutantmentioning

confidence: 73%

Key Enzymes of the Semiphosphorylative Entner-Doudoroff Pathway in the Haloarchaeon Haloferax volcanii: Characterization of Glucose Dehydrogenase, Gluconate Dehydratase, and 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase

et al. 2016

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The halophilic archaeon Haloferax volcanii has been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. So far, the key enzymes of this pathway, glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (KDPGA), have not been characterized, and their functional involvement in glucose degradation has not been demonstrated. Here we report that the genes HVO_1083 and HVO_0950 encode GDH and KDPGA, respectively. The recombinant enzymes show high specificity for glucose and KDPG and did not convert the corresponding C 4 epimers galactose and 2-keto-3-deoxy-6-phosphogalactonate at significant rates. Growth studies of knockout mutants indicate the functional involvement of both GDH and KDPGA in glucose degradation. GAD was purified from H. volcanii, and the encoding gene, gad, was identified as HVO_1488. GAD catalyzed the specific dehydration of gluconate and did not utilize galactonate at significant rates. A knockout mutant of GAD lost the ability to grow on glucose, indicating the essential involvement of GAD in glucose degradation. However, following a prolonged incubation period, growth of the ⌬gad mutant on glucose was recovered. Evidence is presented that under these conditions, GAD was functionally replaced by xylonate dehydratase (XAD), which uses both xylonate and gluconate as substrates. Together, the characterization of key enzymes and analyses of the respective knockout mutants present conclusive evidence for the in vivo operation of the spED pathway for glucose degradation in H. volcanii. IMPORTANCEThe work presented here describes the identification and characterization of the key enzymes glucose dehydrogenase, gluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase and their encoding genes of the proposed semiphosphorylative Entner-Doudoroff pathway in the haloarchaeon Haloferax volcanii. The functional involvement of the three enzymes was proven by analyses of the corresponding knockout mutants. These results provide evidence for the in vivo operation of the semiphosphorylative Entner-Doudoroff pathway in haloarchaea and thus expand our understanding of the unusual sugar degradation pathways in the domain Archaea. Glucose degradation in thermoacidophilic and halophilic archaea has been proposed to proceed via modified versions of the classical Entner-Doudoroff (ED) pathway of bacteria (1, 2) (Fig. 1). For the thermoacidophilic euryarchaeon Picrophilus torridus, a nonphosphorylative ED (npED) pathway was reported (3). Accordingly, glucose is converted to 2-keto-3-deoxygluconate (KDG) via glucose dehydrogenase (GDH) and gluconate dehydratase (GAD). KDG is then cleaved by a KDG aldolase to pyruvate and glyceraldehyde (GA), which is converted to a second molecule of pyruvate via glyceraldehyde dehydrogenase, 2-phosphoglycerate forming glycerate kinase, enolase, and pyruvate kinase. The pathway was proposed to be promiscuous, being involved in the degradation of both D-glucose and its C 4 epimer ...

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Section: Fig 5 Growth Analysis Of the H Volcanii Gad Deletion Mutantmentioning

confidence: 73%

Key Enzymes of the Semiphosphorylative Entner-Doudoroff Pathway in the Haloarchaeon Haloferax volcanii: Characterization of Glucose Dehydrogenase, Gluconate Dehydratase, and 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase

et al. 2016

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“…However, substrate-recognition residues of KDG kinase, which were identified in the S. solfataricus enzymes [42], i.e., Gly34, Tyr90, Tyr106, Arg108, Arg166, Asp258, and Asp294, were entirely conserved in FlKin as Gly34, Tyr89, Tyr104, Arg106, Arg169, Asp280, and Asp317, respectively. FlAld also showed low amino acid identity (22%–25%) with KDPG aldolases from Proteobacteria species such as E. coli (GenBank accession number, WP_000800517) [44,45], Zymomonas mobilis (GenBank accession number, S18559) [44], Pseudomonas putida (GenBank accession number, WP_016501275) [44,46], and S. degradans 2-40 T (GenBank accession number, ABD80644) [25] (Figure 3). Meanwhile, the sequence identities between FlAld and enzymes from other Bacteroidetes species such as G. forsetii KT0803 (GenBank accession number, KT0803), Dokdonia sp.…”

Section: Resultsmentioning

confidence: 99%

“…5H-3-7-4 (GenBank accession number, AEH01606); Dokdo, KDPG aldolase-like protein from Dokdonia sp. MED134 (GenBank accession number, WP_013749799); Grame, KDPG aldolase-like protein from G. forsetii KT0803 (GenBank accession number, CAL66136); Sacch, KDPG aldolase from S. degradans 2-40 T (GenBank accession number, ABD80644) [25]; Esche, KDPG aldolase from E. coli (GenBank accession number, WP_000800517) [44,45]; Zymom, KDPG aldolase from Zymomonas mobilis (GenBank accession number, S18559) [44]; Pseud, KDPG aldolase from Pseudomonas putida (GenBank accession number, WP_016501275) [44,46]. …”

Section: Figurementioning

confidence: 99%

Identification of 2-keto-3-deoxy-d-Gluconate Kinase and 2-keto-3-deoxy-d-Phosphogluconate Aldolase in an Alginate-Assimilating Bacterium, Flavobacterium sp. Strain UMI-01

2017

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Recently, we identified an alginate-assimilating gene cluster in the genome of Flavobacterium sp. strain UMI-01, a member of Bacteroidetes. Alginate lyase genes and a 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH) reductase gene in the cluster have already been characterized; however, 2-keto-3-deoxy-d-gluconate (KDG) kinase and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase genes, i.e., flkin and flald, still remained uncharacterized. The amino acid sequences deduced from flkin and flald showed low identities with those of corresponding enzymes of Saccharophagus degradans 2-40T, a member of Proteobacteria (Kim et al., Process Biochem., 2016). This led us to consider that the DEH-assimilating enzymes of Bacteroidetes species are somewhat deviated from those of Proteobacteria species. Thus, in the present study, we first assessed the characteristics in the primary structures of KDG kinase and KDG aldolase of the strain UMI-01, and then investigated the enzymatic properties of recombinant enzymes, recFlKin and recFlAld, expressed by an Escherichia coli expression system. Multiple-sequence alignment among KDG kinases and KDG aldolases from several Proteobacteria and Bacteroidetes species indicated that the strain UMI-01 enzymes showed considerably low sequence identities (15%–25%) with the Proteobacteria enzymes, while they showed relatively high identities (47%–68%) with the Bacteroidetes enzymes. Phylogenetic analyses for these enzymes indicated the distant relationship between the Proteobacteria enzymes and the Bacteroidetes enzymes, i.e., they formed distinct clusters in the phylogenetic tree. recFlKin and recFlAld produced with the genes flkin and flald, respectively, were confirmed to show KDG kinase and KDPG aldolase activities. Namely, recFlKin produced 1.7 mM KDPG in a reaction mixture containing 2.5 mM KDG and 2.5 mM ATP in a 90-min reaction, while recFlAld produced 1.2 mM pyruvate in the reaction mixture containing 5 mM KDPG at the equilibrium state. An in vitro alginate-metabolizing system constructed from recFlKin, recFlAld, and previously reported alginate lyases and DEH reductase of the strain UMI-01 could convert alginate to pyruvate and glyceraldehyde-3-phosphate with an efficiency of 38%.

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“…They can also catalyze the reversible condensation reaction of the two triose phosphates of pyruvate and GAP into a hexose phosphate. Aldolases can be divided into two types depending on the catalytic mechanism: class I enzymes form a Schiff-base intermediate using the terminal nitrogen atom of the lysine side chain with the carbonyl moiety of the substrate [4][5][6], whereas class II enzymes, without the formation of a Schiff-base intermediate, catalyze the reaction aided by a metal cofactor [7][8][9][10]. Since aldolases can mediate a chemical reaction to introduce a covalent bond between two carbon atoms, many living organisms utilize aldolases for the biosynthesis of a large carbon skeleton.…”

Section: Introductionmentioning

confidence: 99%

“…Bacterial class I KDPG aldolases are proteins composed of ~200 amino acids. Crystal structures of KDPG aldolases from five different microorganisms have revealed a common homotrimeric structure in the crystalline state [4,5,[12][13][14]. Each protomer forms a TIM barrel structure with a central β-barrel of eight β-strands surrounded by the same number of α-helices.…”

Section: Introductionmentioning

confidence: 99%

Structures of Zymomonas 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase with and without a Substrate Analog at the Phosphate-Binding Loop

Seo

Ryu

et al. 2018

Journal of Microbiology and Biotechnology

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2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, which catalyzes aldol cleavage and condensation reactions, has two distinct substrate-binding sites. The substrate-binding mode at the catalytic site and Schiff-base formation have been well studied. However, structural information on the phosphate-binding loop (P-loop) is limited. Zymomonas mobilis KDPG aldolase is one of the aldolases with a wide substrate spectrum. Its structure in complex with the substrate-mimicking 3-phosphoglycerate (3PG) shows that the phosphate moiety of 3PG interacts with the P-loop and a nearby conserved serine residue. 3PG-binding to the P-loop replaces water molecules aligned from the P-loop to the catalytic site, as observed in the apostructure. The extra electron density near the P-loop and comparison with other aldolases suggest the diversity and flexibility of the serine-containing loop among KDPG aldolases. These structural data may help to understand the substrate-binding mode and the broad substrate specificity of the Zymomonas KDPG aldolase.

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Structure of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase fromPseudomonas putida

Cited by 8 publications

References 15 publications

Key Enzymes of the Semiphosphorylative Entner-Doudoroff Pathway in the Haloarchaeon Haloferax volcanii: Characterization of Glucose Dehydrogenase, Gluconate Dehydratase, and 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase

Key Enzymes of the Semiphosphorylative Entner-Doudoroff Pathway in the Haloarchaeon Haloferax volcanii: Characterization of Glucose Dehydrogenase, Gluconate Dehydratase, and 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase

Identification of 2-keto-3-deoxy-d-Gluconate Kinase and 2-keto-3-deoxy-d-Phosphogluconate Aldolase in an Alginate-Assimilating Bacterium, Flavobacterium sp. Strain UMI-01

Structures of Zymomonas 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase with and without a Substrate Analog at the Phosphate-Binding Loop

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