Purification, crystallization and preliminary X-ray diffraction analysis of a novel keto-deoxy-<scp>D</scp>-galactarate (KDG) dehydratase from<i>Agrobacterium tumefaciens</i>

Taberman, Helena; Andberg, Martina; Parkkinen, Tarja; Richard, Peter; Hakulinen, Nina; Koivula, Anu; Rouvinen, Juha

doi:10.1107/s2053230x13031361

Cited by 5 publications

(16 citation statements)

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“…9 The mutagenesis was performed using the Quick Change SiteDirected mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The nucleotide sequence of the mutated gene was confirmed by DNA sequencing.…”

Section: ■ Experimental Proceduresmentioning

confidence: 99%

“…8 The At KDG dehydratase (EC 4.2.1.-) characterized here is supposed to produce from the linear C6 compound α-ketoglutaric semialdehyde a C5 compound (Figure 1). 9 The last step in the oxidative pathway should involve oxidation of α-ketoglutaric semialdehyde to α-ketoglutarate by an α-ketoglutaric semialdehyde (α-KGSA) dehydrogenase (EC 1.2.1.26). 10,11 The α-ketoglutarate then enters the tricarboxylic acid cycle.…”

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confidence: 99%

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Structure and Function of a Decarboxylating Agrobacterium tumefaciens Keto-deoxy-d-galactarate Dehydratase

et al. 2014

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Agrobacterium tumefaciens (At) strain C58 contains an oxidative enzyme pathway that can function on both d-glucuronic and d-galacturonic acid. The corresponding gene coding for At keto-deoxy-d-galactarate (KDG) dehydratase is located in the same gene cluster as those coding for uronate dehydrogenase (At Udh) and galactarolactone cycloisomerase (At Gci) which we have previously characterized. Here, we present the kinetic characterization and crystal structure of At KDG dehydratase, which catalyzes the next step, the decarboxylating hydrolyase reaction of KDG to produce α-ketoglutaric semialdehyde (α-KGSA) and carbon dioxide. The crystal structures of At KDG dehydratase and its complexes with pyruvate and 2-oxoadipic acid, two substrate analogues, were determined to 1.7 Å, 1.5 Å, and 2.1 Å resolution, respectively. Furthermore, mass spectrometry was used to confirm reaction end-products. The results lead us to propose a structure-based mechanism for At KDG dehydratase, suggesting that while the enzyme belongs to the Class I aldolase protein family, it does not follow a typical retro-aldol condensation mechanism.

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Section: ■ Experimental Proceduresmentioning

confidence: 99%

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confidence: 99%

Structure and Function of a Decarboxylating Agrobacterium tumefaciens Keto-deoxy-d-galactarate Dehydratase

et al. 2014

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“…To find the galacturonic acid transporter, the genome was scanned for genes known to be involved in galacturonic acid catabolism, searching for nearby putative transporter candidates. This search revealed a region on the linear chromosome encoding proteins previously identified as being involved in galacturonic acid catabolism (23)(24)(25)(26)(27)(28)(29). Nearby are three gene loci (Atu3135, Atu3136, and Atu3137) that encode proteins with homologs to the TRAP family transporters.…”

Section: Resultsmentioning

confidence: 99%

“…Galactarolactone cycloisomerase (encoded by gene locus Atu3139) (27) catalyzes the ring opening of galactaro-1,4-lactone to form 3-deoxy-2-keto-L-threo-hexarate, which is converted to ␣-ketoglutarate semialdehyde through the function of a keto-deoxy-D-galactarate (KDG) dehydratase (encoded by gene locus Atu3140) (28,29). In the last step, a dehydrogenase oxidizes ␣-ketoglutarate semialdehyde to form ␣-ketoglutarate, which is an intermediate in the citric acid cycle (30).…”

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Involvement of Agrobacterium tumefaciens Galacturonate Tripartite ATP-Independent Periplasmic (TRAP) Transporter GaaPQM in Virulence Gene Expression

Zhao

Binns

2016

Appl Environ Microbiol

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Monosaccharides capable of serving as nutrients for the soil bacterium Agrobacterium tumefaciens are also inducers of the vir regulon present in the tumor-inducing (Ti) plasmid of this plant pathogen. One such monosaccharide is galacturonate, the predominant monomer of pectin found in plant cell walls. This ligand is recognized by the periplasmic sugar binding protein ChvE, which interacts with the VirA histidine kinase that controls vir gene expression. Although ChvE is also a member of the ChvEMmsAB ABC transporter involved in the utilization of many neutral sugars, it is not involved in galacturonate utilization. In this study, a putative tripartite ATP-independent periplasmic (TRAP) transporter, GaaPQM, is shown to be essential for the utilization of galacturonic acid; we show that residue R169 in the predicted sugar binding site of the GaaP is required for activity. The gene upstream of gaaPQM (gaaR) encodes a member of the GntR family of regulators. GaaR is shown to repress the expression of gaaPQM, and the repression is relieved in the presence of the substrate for GaaPQM. Moreover, GaaR is shown to bind putative promoter regions in the sequences required for galacturonic acid utilization. Finally, A. tumefaciens strains carrying a deletion of gaaPQM are more sensitive to galacturonate as an inducer of vir gene expression, while the overexpression of gaaPQM results in strains being less sensitive to this vir inducer. This supports a model in which transporter activity is crucial in ensuring that vir gene expression occurs only at sites of high ligand concentration, such as those at a plant wound site.

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“…Only little attention has been devoted towards this enzyme even though it catalyses a very interesting reaction. Just recently, the crystal structure for KdgD from A. tumefaciens was solved [22] in parallel with investigations to gain a deeper understanding of the catalytic mechanism, which led to the identification of catalytically relevant amino acids [23]. …”

Section: Introductionmentioning

confidence: 99%

Identification and characterization of two new 5-keto-4-deoxy-D-Glucarate Dehydratases/Decarboxylases

et al. 2016

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BackgroundHexuronic acids such as D-galacturonic acid and D-glucuronic acid can be utilized via different pathways within the metabolism of microorganisms. One representative, the oxidative pathway, generates α-keto-glutarate as the direct link entering towards the citric acid cycle. The penultimate enzyme, keto-deoxy glucarate dehydratase/decarboxylase, catalyses the dehydration and decarboxylation of keto-deoxy glucarate to α-keto-glutarate semialdehyde. This enzymatic reaction can be tracked continuously by applying a pH-shift assay.ResultsTwo new keto-deoxy glucarate dehydratases/decarboxylases (EC 4.2.1.41) from Comamonas testosteroni KF-1 and Polaromonas naphthalenivorans CJ2 were identified and expressed in an active form using Escherichia coli ArcticExpress(DE3). Subsequent characterization concerning K m, k cat and thermal stability was conducted in comparison with the known keto-deoxy glucarate dehydratase/decarboxylase from Acinetobacter baylyi ADP1. The kinetic constants determined for A. baylyi were K m 1.0 mM, k cat 4.5 s−1, for C. testosteroni K m 1.1 mM, k cat 3.1 s−1, and for P. naphthalenivorans K m 1.1 mM, k cat 1.7 s−1. The two new enzymes had a slightly lower catalytic activity (increased K m and a decreased k cat) but showed a higher thermal stability than that of A. baylyi. The developed pH-shift assay, using potassium phosphate and bromothymol blue as the pH indicator, enables a direct measurement. The use of crude extracts did not interfere with the assay and was tested for wild-type landscapes for all three enzymes.ConclusionsBy establishing a pH-shift assay, an easy measurement method for keto-deoxy glucarate dehydratase/decarboxylase could be developed. It can be used for measurements of the purified enzymes or using crude extracts. Therefore, it is especially suitable as the method of choice within an engineering approach for further optimization of these enzymes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0308-3) contains supplementary material, which is available to authorized users.

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Purification, crystallization and preliminary X-ray diffraction analysis of a novel keto-deoxy-D-galactarate (KDG) dehydratase fromAgrobacterium tumefaciens

Cited by 5 publications

References 25 publications

Structure and Function of a Decarboxylating Agrobacterium tumefaciens Keto-deoxy-d-galactarate Dehydratase

Structure and Function of a Decarboxylating Agrobacterium tumefaciens Keto-deoxy-d-galactarate Dehydratase

Involvement of Agrobacterium tumefaciens Galacturonate Tripartite ATP-Independent Periplasmic (TRAP) Transporter GaaPQM in Virulence Gene Expression

Identification and characterization of two new 5-keto-4-deoxy-D-Glucarate Dehydratases/Decarboxylases

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