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Cloning and expression of 3-deoxy-d-manno-oct-2-ulosonic acid ?-ketoside hydrolase from oyster hepatopancreas†.

ABSTRACT: We have previously reported that oyster hepatopancreas contained three unusual ?-ketoside hydrolases: (i) a 3-deoxy-d-manno-oct-2-ulosonic acid ?-ketoside hydrolase (?-Kdo-ase), (ii) a 3-deoxy-D-glycero-D-galacto-non-2-ulosonic acid ?-ketoside hydrolase and (iii) a bifunctional ketoside hydrolase capable of cleaving both the ?-ketosides of Kdn and Neu5Ac (Kdn-sialidase). After completing the purification of Kdn-sialidase, we proceeded to clone the gene encoding this enzyme. Unexpectedly, we found that instead of expressing Kdn-sialidase, our cloned gene expressed ?-Kdo-ase activity. The full-length gene, consisting of 1176-bp (392 amino acids, Mr 44,604), expressed an active recombinant ?-Kdo-ase (R-?-Kdo-ase) in yeast and CHO-S cells, but not in various Escherichia coli strains. The deduced amino acid sequence contains two Asp boxes (S(277)PDDGKTW and S(328)TDQGKTW) commonly found in sialidases, but is devoid of the signature FRIP-motif of sialidase. The R-?-Kdo-ase effectively hydrolyzed the Kdo in the core-oligosaccharide of the structurally defined lipopolysaccharide (LPS), Re-LPS (Kdo(2)-Lipid A) from Salmonella minnesota R595 and E. coli D31m4. However, Rd-LPS from S. minnesota R7 that contained an extra outer core phosphorylated heptose was only slowly hydrolyzed. The complex type LPS from Neisseria meningitides A1 and M992 that contained extra 5-6 sugar units at the outer core were refractory to R-?-Kdo-ase. This R-?-Kdo-ase should become useful for studying the structure and function of Kdo-containing glycans.

SUBMITTER: Nakagawa T

PROVIDER: S-EPMC4634316 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Cloning and expression of 3-deoxy-d-manno-oct-2-ulosonic acid α-ketoside hydrolase from oyster hepatopancreas†.

Nakagawa Tetsuto T Shimada Yoshimi Y Pavlova Nadejda V NV Li Su-Chen SC Li Yu-Teh YT

Glycobiology 20150911 12

We have previously reported that oyster hepatopancreas contained three unusual α-ketoside hydrolases: (i) a 3-deoxy-d-manno-oct-2-ulosonic acid α-ketoside hydrolase (α-Kdo-ase), (ii) a 3-deoxy-D-glycero-D-galacto-non-2-ulosonic acid α-ketoside hydrolase and (iii) a bifunctional ketoside hydrolase capable of cleaving both the α-ketosides of Kdn and Neu5Ac (Kdn-sialidase). After completing the purification of Kdn-sialidase, we proceeded to clone the gene encoding this enzyme. Unexpectedly, we foun ...[more]

PMID: 26362869

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Project description:Metabolic labeling paired with click chemistry is a powerful approach for selectively imaging the surfaces of diverse bacteria. Herein, we explored the feasibility of labeling the lipopolysaccharide (LPS) of Myxococcus xanthus-a Gram-negative predatory social bacterium known to display complex outer membrane (OM) dynamics-via growth in the presence of distinct azido (-N3) analogues of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo). Determination of the LPS carbohydrate structure from strain DZ2 revealed the presence of one Kdo sugar in the core oligosaccharide, modified with phosphoethanolamine. The production of 8-azido-8-deoxy-Kdo (8-N3-Kdo) was then greatly improved over previous reports via optimization of the synthesis of its 5-azido-5-deoxy-d-arabinose precursor to yield gram amounts. The novel analogue 7-azido-7-deoxy-Kdo (7-N3-Kdo) was also synthesized, with both analogues capable of undergoing in vitro strain-promoted azide-alkyne cycloaddition (SPAAC) "click" chemistry reactions. Slower and faster growth of M. xanthus was displayed in the presence of 8-N3-Kdo and 7-N3-Kdo (respectively) compared to untreated cells, with differences also seen for single-cell gliding motility and type IV pilus-dependent swarm community expansion. While the surfaces of 8-N3-Kdo-grown cells were fluorescently labeled following treatment with dibenzocyclooctyne-linked fluorophores, the surfaces of 7-N3-Kdo-grown cells could not undergo fluorescent tagging. Activity analysis of the KdsB enzyme required to activate Kdo prior to its integration into nascent LPS molecules revealed that while 8-N3-Kdo is indeed a substrate of the enzyme, 7-N3-Kdo is not. Though a lack of M. xanthus cell aggregation was shown to expedite growth in liquid culture, 7-N3-Kdo-grown cells did not manifest differences in intrinsic clumping relative to untreated cells, suggesting that 7-N3-Kdo may instead be catabolized by the cells. Ultimately, these data provide important insights into the synthesis and cellular processing of valuable metabolic labels and establish a basis for the elucidation of fundamental principles of OM dynamism in live bacterial cells.

Dataset Information

Cloning and expression of 3-deoxy-d-manno-oct-2-ulosonic acid ?-ketoside hydrolase from oyster hepatopancreas†.

Publications

Cloning and expression of 3-deoxy-d-manno-oct-2-ulosonic acid α-ketoside hydrolase from oyster hepatopancreas†.

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