Biochemical Characterization of the CDP-D-Arabinitol Biosynthetic Pathway in Streptococcus pneumoniae 17F

CPS (capsular polysaccharide) is a major virulence factor in Streptococcus pneumoniae. Biosynthesis of CPS RU (repeat unit) proceeds by sequential transfer of sugar residues from the appropriate sugar donor to an activated lipid carrier by committed GTs (glycosyltransferases). While the nucleotide sequence of many cps loci is already known, the real substrate specificity of the hypothetical GTs, as well as the sequence of sugar addition is unclear. In the present paper, we report the biochemical characterization of one α-galactosyltransferase, WciS (Cap8H), a member of GT family 4. This enzyme is implicated in the tetrasaccharide RU biosynthetic pathway of Strep. pneumoniae CPS 8 ([→4)-α-D-Glcp-(1→4)-α-D-Galp-(1→4)-β-D-GlcAp-(1→4)-β-D-Glcp-(1→]n). Expression of WciS–His6 in Escherichia coli BL21 (DE3) strains or BL21 (DE3)/ΔgalU strain resulted in synthesis of a 39 kDa membrane-associated protein identified by N-terminal sequencing and recognized by anti-His6-tag antibody. This protein was capable of adding a galactose residue cellobiuronic acid [β-D-GlcAp-(1→4)-D-Glcp]-pyrophosphate-polyprenol from UDP-Gal. The newly added galactose residue is removed by α-galactosidase, indicating that WciS is a retaining GT. Our results suggest that WciS catalyses the addition of the third sugar residue of the CPS 8 RU. The recombinant WciS–His6 was solubilized and purified as a soluble multimer, opening the way for structural studies.

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Biochemical Characterization of the First Essential Two-Component Signal Transduction System from Staphylococcus aureus and Streptococcus pneumoniae

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000071077 ◽

2003 ◽

Vol 5 (4) ◽

pp. 252-260 ◽

Cited By ~ 26

Author(s):

Valerie A. Clausen ◽

Weonhye Bae ◽

John Throup ◽

Martin K.R. Burnham ◽

Martin Rosenberg ◽

...

Keyword(s):

Signal Transduction ◽

Staphylococcus Aureus ◽

Streptococcus Pneumoniae ◽

Biochemical Characterization ◽

Signal Transduction System ◽

Two Component ◽

Two Component Signal Transduction

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Discovery and Biocatalytic Application of a PLP-Dependent Amino Acid γ-Substitution Enzyme that Catalyzes C-C Bond Formation

10.26434/chemrxiv.12052410 ◽

2020 ◽

Author(s):

Mengbin Chen ◽

Chun-Ting Liu ◽

Yi Tang

Keyword(s):

Amino Acids ◽

Biosynthetic Pathway ◽

Pyridoxal Phosphate ◽

Biochemical Characterization ◽

Indole Alkaloid ◽

Building Blocks ◽

Nucleophilic Attack ◽

Keto Esters ◽

Key Enzyme

Pyridoxal phosphate (PLP)-dependent enzymes can catalyze various transformations of amino acids at alpha, beta, and gamma positions. These versatile enzymes are prominently involved in the biosynthesis of nonproteinogenic amino acids as building blocks of natural products, and are attractive biocatalysts. Here, we report the discovery of a two-step enzymatic synthesis of (2S, 6S)-6-methyl pipecolate 1, from the biosynthetic pathway of indole alkaloid citrinadin. The key enzyme CndF is PLP-dependent and catalyzes synthesis of (S)-2-amino-6-oxoheptanoate 3 that is in equilibrium with the cyclic Schiff base. The second enzyme CndE is a stereoselective imine reductase that gives 1. Biochemical characterization of CndF showed this enzyme performs gamma-elimination of O-acetyl L-homoserine to generate the vinylglycine ketimine, which is subjected to nucleophilic attack by acetoacetate to form the new Cgamma-Cdelta bond in 3 and complete the gamma-substitution reaction. CndF displays substrate promiscuity towards different beta-keto carboxylate and esters. Using a recombinant Aspergillus strain expressing CndF and CndE, feeding various alkyl-beta-keto esters led to the biosynthesis of 6-substituted L-pipecolates. The discovery of CndF expands the repertoire of reactions that can be catalyzed by PLP-dependent enzymes.

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Characterization of an Escherichia coli K12 mutant that is sensitive to chlorate when grown aerobically

Biochemical Journal ◽

10.1042/bj1760553 ◽

1978 ◽

Vol 176 (2) ◽

pp. 553-561 ◽

Cited By ~ 17

Author(s):

G Giordano ◽

L Grillet ◽

R Rosset ◽

J H Dou ◽

E Azoulay ◽

...

Keyword(s):

Escherichia Coli ◽

Biosynthetic Pathway ◽

Formate Dehydrogenase ◽

Biochemical Characterization ◽

Reductase Activity ◽

Growth Conditions ◽

Aerobic Growth ◽

Benzyl Viologen ◽

Genetic Lesion

Escherichia coli can normally grow aerobically in the presence of chlorate; however, mutants can be isolated that can no longer grow under these conditions. We present here the biochemical characterization of one such mutant and show that the primary genetic lesion occurs in the ubiquinone-8-biosynthetic pathway. As a consequence of this, under aerobic growth conditions the mutant is apparently unable to synthesize formate dehydrogenase, but can synthesize a Benzyl Viologen-dependent nitrate reductase activity. The nature of this activity is discussed.

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Characterization of the CDP-2-Glycerol Biosynthetic Pathway in Streptococcus pneumoniae

Journal of Bacteriology ◽

10.1128/jb.00561-10 ◽

2010 ◽

Vol 192 (20) ◽

pp. 5506-5514 ◽

Cited By ~ 8

Author(s):

Quan Wang ◽

Yanli Xu ◽

Andrei V. Perepelov ◽

Wei Xiong ◽

Dongmei Wei ◽

...

Keyword(s):

Mass Spectrometry ◽

Streptococcus Pneumoniae ◽

Biosynthetic Pathway ◽

Resonance Spectroscopy ◽

Ionization Mass ◽

Enzyme Substrate ◽

Effects Of Temperature ◽

First Time ◽

Novel Products

ABSTRACT Capsule polysaccharide (CPS) plays an important role in the virulence of Streptococcus pneumoniae and is usually used as the pneumococcal vaccine target. Glycerol-2-phosphate is found in the CPS of S. pneumoniae types 15A and 23F and is rarely found in the polysaccharides of other bacteria. The biosynthetic pathway of the nucleotide-activated form of glycerol-2-phosphate (NDP-2-glycerol) has never been identified. In this study, three genes (gtp1, gtp2, and gtp3) from S. pneumoniae 23F that have been proposed to be involved in the synthesis of NDP-2-glycerol were cloned and the enzyme products were expressed, purified, and assayed for their respective activities. Capillary electrophoresis was used to detect novel products from the enzyme-substrate reactions, and the structure of the product was elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. Gtp1 was identified as a reductase that catalyzes the conversion of 1,3-dihydroxyacetone to glycerol, Gtp3 was identified as a glycerol-2-phosphotransferase that catalyzes the conversion of glycerol to glycerol-2-phosphate, and Gtp2 was identified as a cytidylyltransferase that transfers CTP to glycerol-2-phosphate to form CDP-2-glycerol as the final product. The kinetic parameters of Gtp1 and Gtp2 were characterized in depth, and the effects of temperature, pH, and cations on these two enzymes were analyzed. This is the first time that the biosynthetic pathway of CDP-2-glycerol has been identified biochemically; this pathway provides a method to enzymatically synthesize this compound.

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Physical and biochemical characterization of the cloned LYS5 gene required for ?-aminoadipate reductase activity in the lysine biosynthetic pathway of Saccharomyces cerevisiae

Current Genetics ◽

10.1007/bf00318647 ◽

1992 ◽

Vol 21 (1) ◽

pp. 13-16 ◽

Cited By ~ 3

Author(s):

Sudha Rajnarayan ◽

Jack C. Vaughn ◽

J. K. Bhattacharjee

Keyword(s):

Saccharomyces Cerevisiae ◽

Biosynthetic Pathway ◽

Biochemical Characterization ◽

Reductase Activity ◽

Lys5 Gene

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Reconstitution and Biochemical Characterization of a New Pyridoxal-5‘-Phosphate Biosynthetic Pathway

Journal of the American Chemical Society ◽

10.1021/ja042792t ◽

2005 ◽

Vol 127 (11) ◽

pp. 3682-3683 ◽

Cited By ~ 81

Author(s):

Kristin E. Burns ◽

Yun Xiang ◽

Cynthia L. Kinsland ◽

Fred W. McLafferty ◽

Tadhg P. Begley

Keyword(s):

Biosynthetic Pathway ◽

Biochemical Characterization

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Characterization of a SAM-dependent fluorinase from a latent biosynthetic pathway for fluoroacetate and 4-fluorothreonine formation in Nocardia brasiliensis

F1000Research ◽

10.12688/f1000research.3-61.v1 ◽

2014 ◽

Vol 3 ◽

pp. 61 ◽

Cited By ~ 11

Author(s):

Yaya Wang ◽

Zixin Deng ◽

Xudong Qu

Keyword(s):

Chemical Synthesis ◽

Kinetic Parameters ◽

Sequence Comparison ◽

Biosynthetic Pathway ◽

Biochemical Characterization ◽

Inorganic Fluoride ◽

Nocardia Brasiliensis ◽

Loop Region ◽

Streptomyces Cattleya

Fluorination has been widely used in chemical synthesis, but is rare in nature. The only known biological fluorination scope is represented by theflpathway fromStreptomyces cattleyathat produces fluoroacetate (FAc) and 4-fluorothreonine (4-FT). Here we report the identification of a novel pathway for FAc and 4-FT biosynthesis from the actinomycetoma-causing pathogenNocardia brasiliensisATCC 700358. The new pathway shares overall conservation with theflpathway inS. cattleya. Biochemical characterization of the conserved domains revealed a novel fluorinase NobA that can biosynthesize 5’-fluoro-5’-deoxyadenosine (5’-FDA) from inorganic fluoride andS-adenosyl-l-methionine (SAM). The NobA shows similar halide specificity and characteristics to the fluorination enzyme FlA of theflpathway. Kinetic parameters for fluoride (Km4153 μM,kcat0.073 min-1) and SAM (Km416 μM,kcat0.139 min-1) have been determined, revealing that NobA is slightly (2.3 fold) slower than FlA. Upon sequence comparison, we finally identified a distinct loop region in the fluorinases that probably accounts for the disparity of fluorination activity.

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