The First Crystal Structure of Archaeal Aldolase

Sakuraba, Haruhiko; Tsuge, Hideaki; Shimoya, I.; Kawakami, Retsuo; Goda, Shuichiro; Kawarabayasi, Yutaka; Katunuma, Nobuhiko; Ago, Hideo; Miyano, Masashi; Ohshima, Toshihisa

doi:10.1074/jbc.m212449200

Cited by 43 publications

(21 citation statements)

References 39 publications

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“…Interestingly, the dimer interface of Tg DPA forms around the flipped out N-terminal helix (Fig. 2B), similar to the dimerization interface predicted for Py DERA [46], but significantly different than the dimer and tetramer-forming interfaces observed for other dR5P aldolases [44, 47, 48]. Additionally, no salt bridges and only one hydrogen bond were present at the interface consistent with a largely hydrophobic-based assembly.…”

Section: Resultssupporting

confidence: 60%

Structural and Functional Divergence of the Aldolase Fold in Toxoplasma gondii

Tonkin

Halavaty

Ramaswamy

et al. 2015

Journal of Molecular Biology

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Parasites of the phylum Apicomplexa are highly successful pathogens of humans and animals worldwide. As obligate intracellular parasites, they have significant energy requirements for invasion and gliding motility that are supplied by various metabolic pathways. Aldolases have emerged as key enzymes involved in these pathways, and all apicomplexans express one or both of fructose 1,6-bisphosphate (F16BP) aldolase and 2-deoxyribose 5-phosphate (dR5P) aldolase (DERA). Intriguingly, Toxoplasma gondii, a highly successful apicomplexan parasite, expresses F16BP aldolase (TgALD1), d5RP aldolase (TgDERA), and a divergent dR5P aldolase-like protein (TgDPA) exclusively in the latent bradyzoite stage. While the importance of TgALD1 in glycolysis is well established and TgDERA is also likely to be involved in parasite metabolism, the detailed function of TgDPA remains elusive. To gain mechanistic insight into the function of different T. gondii aldolases, we first determined the crystal structures of TgALD1 and TgDPA. Structural analysis revealed that both aldolases adopt a TIM barrel fold accessorized with divergent secondary structure elements. Structural comparison of TgALD1 and TgDPA with members of their respective enzyme families revealed that while the active site residues are conserved in TgALD1, key catalytic residues are absent in TgDPA. Consistent with this observation, biochemical assays showed that while TgALD1 was active on F16BP, TgDPA was inactive on dR5P. Intriguingly, both aldolases are competent to bind polymerized actin in vitro. Altogether, structural and biochemical analyses of T. gondii aldolase and aldolase-like proteins reveal diverse functionalization of the classic TIM barrel aldolase fold.

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Section: Resultssupporting

confidence: 60%

Structural and Functional Divergence of the Aldolase Fold in Toxoplasma gondii

Tonkin

Halavaty

Ramaswamy

et al. 2015

Journal of Molecular Biology

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“…An enlargement of the oligomatic interface is also shown to be important for the thermostability, for instance, in 2-deoxy-D-ribose-5-phosphate aldolase. 19 However, we found that the dimer contact area of DHQS is independent of the living temperature. From these results, the dimeric state of DHQS might not contribute to the thermostability, probably because the wellconserved dimeric structure itself is relevant to the catalytic function of this enzyme.…”

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confidence: 38%

Crystal structure of dehydroquinate synthase from Thermus thermophilus HB8 showing functional importance of the dimeric state

et al. 2004

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Introduction. Dehydroquinate synthase (DHQS) is a ␤-nicotinamide adenine dinucleotide (NAD ϩ )-dependent metalloenzyme that converts 3-deoxy-D-arabino-heptulosonate-7-phosphate to dehydroquinate in the shikimate pathway of bacteria, microbial eukaryotes, and plants. 1 This enzyme is expected to be one of the targets for novel antifungal and antibacterial drug designs, 2,3 because the shikimate pathway is absent in mammals, and DHQS is required for pathogenic virulence.4 DHQS is known to have a multistep mechanism, where a single active site catalyzes 5 sequential reactions involving alcohol oxidation, phosphate elimination, carbonyl reduction, ring opening, and intramolecular aldol condensation. 5,6 The crystal structure of DHQS from Aspergillus nidulans (AnDHQS) in complex with substrates and metal ions 7,8 elucidated important residues in the active site and ligand-induced intersubunit orientational changes that provide a potential reaction mechanism. The AnDHQS structures also revealed their dimeric state in crystal form, although its biological significance was not fully discussed. This time, we have solved the crystal structure of DHQS from Thermus thermophilus HB8 (TtDHQS) at 1.8 Å resolution, which is the first crystal structure of this enzyme from thermophilic organisms. We show that TtDHQS is present as a homodimer in crystal form, as well as in solution. In this article, we present the structural comparison between AnDHQS and TtDHQS, providing insight into the biological significance of the dimeric state of this enzyme. Materials and Methods. Sample preparation:The expression plasmid pET-11a, which carries a gene encoding TtDHQS (residues 1-348) from the T. thermophilus HB8 genome, 9 was prepared by the RIKEN Structurome Group. Escherichia coli BL21 (DE3) cells were transformed with the expression plasmid and grown at 37°C in LuriaBertani (LB) medium containing 50 g/mL ampicillin for 20 h. The cells were harvested by centrifugation at 6500 rpm for 5 min, suspended in 20 mM Tris-HCl, pH 8.0 (buffer A), containing 0.5 M NaCl and 5 mM 2-mercaptoethanol, and disrupted by sonication. The supernatant was heated at 70°C for 10 min. After the heat treatment, cell debris and denatured proteins were removed by centrifugation (14,000 rpm, 30 min), and the supernatant was used as the crude extract for purification. The crude extract was desalted using a HiPrep 26/10 desalting column (Amersham-Biosciences) and applied onto a SuperQ TOYOPEAL 650 M column (Tosoh) equilibrated with buffer A. Proteins were eluted with a linear gradient of 0 -0.4 M NaCl. After the buffer replacement with 20 mM 2-Morpholinoethanesulfonic acid (MES)-NaOH pH 6.0 (buffer B), the fraction containing TtDHQS was subjected to a RESOURCE S column (Amersham-Biosciences) equilibrated with buffer B, and eluted with a linear gradient of 0 -0.4 M NaCl in buffer B. After the buffer replacement with 10 mM phosphate-NaOH, pH 7.0, the fraction containing TtDHQS was applied onto a Bio-Scale CHT-20-I column (BIO-RAD) equilibrated with the same buffer. Prot...

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“…EA01524.86509.1137 c Maximal activity at 200 mM(Kim et al 2009) Paenibacillus sp . EA00124.565014562 c n.s.(Kim et al 2010) Rhodococcus erythropolis 22.9725 e 4.8417 c Half-life = 64.4 min300 mM 25 °C(Kullartz and Pietruszka 2012) Haemophilus influenza 23.67.5400.1470.42 b Maximal activity at 300 mM(Woo et al 2014) Staphylococcus epidermidis 29.2n.s.67.1 c 11.3% retention of activity2 h 300 mM 25 °C(Fei et al 2015) Lactobacillus brevis ECU8302n.s.6403.34102 b Half-life = 37.3 min300 mM 25 °C(Jiao et al 2015)Extremophiles (hyperthermophilic unless specified) Aeropyrum pernix 24.56.5n.s.0.057**4.5 c **n.s.(Sakuraba et al 2003) Thermococcus kodakaraensis 24.54950.81***285 b ***n.s.(Rashid et al 2004) Pyrobaculum aerophilum 24.56n.s.0.0660.25 c 53% retention of activity20 h 300 mM 25 °C(Sakuraba et al 2007) Thermotoga maritima 27.86.5n.s.0.021 c 46% retention of activity20 h 300 mM 25 °C(Sakuraba et al 2007) Hyperthermus butylicus 26.45.5800.150.5 c 75% retention of activity8 h 300 mM 25 °C(Wang et al 2010) Aciduliprofundum boonei 26.67800.12n.s.70% retention of activity4 h 250 mM 25 °C(Yin et al 201...…”

Section: Substrate Specificitymentioning

confidence: 99%

2-Deoxy-d-ribose-5-phosphate aldolase (DERA): applications and modifications

Haridas

Abdelraheem

Hanefeld

2018

Appl Microbiol Biotechnol

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2-Deoxy-d-ribose-5-phosphate aldolase (DERA) is a class I aldolase that offers access to several building blocks for organic synthesis. It catalyzes the stereoselective C–C bond formation between acetaldehyde and numerous other aldehydes. However, the practical application of DERA as a biocatalyst is limited by its poor tolerance towards industrially relevant concentrations of aldehydes, in particular acetaldehyde. Therefore, the development of proper experimental conditions, including protein engineering and/or immobilization on appropriate supports, is required. The present review is aimed to provide a brief overview of DERA, its history, and progress made in understanding the functioning of the enzyme. Furthermore, the current understanding regarding aldehyde resistance of DERA and the various optimizations carried out to modify this property are discussed.

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The First Crystal Structure of Archaeal Aldolase

Cited by 43 publications

References 39 publications

Structural and Functional Divergence of the Aldolase Fold in Toxoplasma gondii

Structural and Functional Divergence of the Aldolase Fold in Toxoplasma gondii

Crystal structure of dehydroquinate synthase from Thermus thermophilus HB8 showing functional importance of the dimeric state

2-Deoxy-d-ribose-5-phosphate aldolase (DERA): applications and modifications

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