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Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase from Streptococcus suis serotype 2.

ABSTRACT: 2-Keto-3-deoxy-6-phosphogluconate (KDPG) adolase from pathogenic Streptococcus suis serotype 2 was crystallized using the hanging-drop vapour-diffusion method at 291 K. X-ray diffraction data were collected to 2.8 A resolution. The crystal belonged to space group R32, with unit-cell parameters a = b = 126.4, c = 415.9 A, alpha = beta = 90, gamma = 120 degrees . Assuming the presence of six molecules in the asymmetric unit gave a V(M) value of 2.32 A(3) Da(-1) and a solvent content of 47.12%.

SUBMITTER: Zhang Q

PROVIDER: S-EPMC2581687 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase from Streptococcus suis serotype 2.

Zhang Qiangmin Q Gao Feng F Qi Jianxun J Cheng Hao H Liu Yiwei Y Gao George F GF

Acta crystallographica. Section F, Structural biology and crystallization communications 20081025 Pt 11

2-Keto-3-deoxy-6-phosphogluconate (KDPG) adolase from pathogenic Streptococcus suis serotype 2 was crystallized using the hanging-drop vapour-diffusion method at 291 K. X-ray diffraction data were collected to 2.8 A resolution. The crystal belonged to space group R32, with unit-cell parameters a = b = 126.4, c = 415.9 A, alpha = beta = 90, gamma = 120 degrees . Assuming the presence of six molecules in the asymmetric unit gave a V(M) value of 2.32 A(3) Da(-1) and a solvent content of 47.12%. ...[more]

PMID: 18997324

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Project description: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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Dataset Information

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase from Streptococcus suis serotype 2.

Publications

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase from Streptococcus suis serotype 2.

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