Mass spectrometric analysis of chitin oligosaccharides produced by Rhizobium NodC protein in Escherichia coli.

ABSTRACT In vivo reconstitution of the TDP-l-megosamine pathway from the megalomicin gene cluster of Micromonospora megalomicea was accomplished by the heterologous expression of its biosynthetic genes in Escherichia coli. Mass spectrometric analysis of the TDP-sugar intermediates produced from operons containing different sets of genes showed that the production of TDP-l-megosamine from TDP-4-keto-6-deoxy-d-glucose requires only five biosynthetic steps, catalyzed by MegBVI, MegDII, MegDIII, MegDIV, and MegDV. Bioconversion studies demonstrated that the sugar transferase MegDI, along with the helper protein MegDVI, catalyzes the transfer of l-megosamine to either erythromycin C or erythromycin D, suggesting two possible routes for the production of megalomicin A. Analysis in vivo of the hydroxylation step by MegK indicated that erythromycin C is the intermediate of megalomicin A biosynthesis.

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Serendipitous crystallization and structure determination of cyanase (CynS) fromSerratia proteamaculans

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15004902 ◽

2015 ◽

Vol 71 (4) ◽

pp. 471-476 ◽

Cited By ~ 4

Author(s):

Agata Butryn ◽

Gabriele Stoehr ◽

Christian Linke-Winnebeck ◽

Karl-Peter Hopfner

Keyword(s):

Escherichia Coli ◽

Carbon Dioxide ◽

Structure Determination ◽

Active Site ◽

Mass Spectrometric ◽

Mass Spectrometric Analysis ◽

Spectrometric Analysis ◽

Conserved Residues ◽

Serratia Proteamaculans

Cyanate hydratase (CynS) catalyzes the decomposition of cyanate and bicarbonate into ammonia and carbon dioxide. Here, the serendipitous crystallization of CynS fromSerratia proteamaculans(SpCynS) is reported. SpCynS was crystallized as an impurity and its identity was determined using mass-spectrometric analysis. The crystals belonged to space groupP1 and diffracted to 2.1 Å resolution. The overall structure of SpCynS is very similar to a previously determined structure of CynS fromEscherichia coli. Density for a ligand bound to the SpCynS active site was observed, but could not be unambiguously identified. Additionally, glycerol molecules bound at the entry to the active site of the enzyme indicate conserved residues that might be important for the trafficking of substrates and products.

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Characterization of cardiolipin from Escherichia coli by electrospray ionization with multiple stage quadrupole ion-trap mass spectrometric analysis of [M−2H+Na]− ions

Journal of the American Society for Mass Spectrometry ◽

10.1016/j.jasms.2005.11.019 ◽

2006 ◽

Vol 17 (3) ◽

pp. 420-429 ◽

Cited By ~ 55

Author(s):

Fong -Fu Hsu ◽

John Turk

Keyword(s):

Escherichia Coli ◽

Electrospray Ionization ◽

Ion Trap ◽

Quadrupole Ion Trap ◽

Mass Spectrometric ◽

Mass Spectrometric Analysis ◽

Spectrometric Analysis ◽

Na Ions

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Characterization of the Activities of the CpeY, CpeZ, and CpeS Bilin Lyases in Phycoerythrin Biosynthesis in Fremyella diplosiphon Strain UTEX 481

Journal of Biological Chemistry ◽

10.1074/jbc.m111.284281 ◽

2011 ◽

Vol 286 (41) ◽

pp. 35509-35521 ◽

Cited By ~ 28

Author(s):

Avijit Biswas ◽

M. Nazim Boutaghou ◽

Richard M. Alvey ◽

Christina M. Kronfel ◽

Richard B. Cole ◽

...

Keyword(s):

Escherichia Coli ◽

Light Harvesting ◽

Fluorescence Emission ◽

Green Light ◽

Mass Spectrometric ◽

Mass Spectrometric Analysis ◽

Spectrometric Analysis ◽

Site Specific ◽

Fremyella Diplosiphon

When grown in green light, Fremyella diplosiphon strain UTEX 481 produces the red-colored protein phycoerythrin (PE) to maximize photosynthetic light harvesting. PE is composed of two subunits, CpeA and CpeB, which carry two and three phycoerythrobilin (PEB) chromophores, respectively, that are attached to specific Cys residues via thioether linkages. Specific bilin lyases are hypothesized to catalyze each PEB ligation. Using a heterologous, coexpression system in Escherichia coli, the PEB ligation activities of putative lyase subunits CpeY, CpeZ, and CpeS were tested on the CpeA and CpeB subunits from F. diplosiphon. Purified His6-tagged CpeA, obtained by coexpressing cpeA, cpeYZ, and the genes for PEB synthesis, had absorbance and fluorescence emission maxima at 566 and 574 nm, respectively. CpeY alone, but not CpeZ, could ligate PEB to CpeA, but the yield of CpeA-PEB was lower than achieved with CpeY and CpeZ together. Studies with site-specific variants of CpeA(C82S and C139S), together with mass spectrometric analysis of trypsin-digested CpeA-PEB, revealed that CpeY/CpeZ attached PEB at Cys82 of CpeA. The CpeS bilin lyase ligated PEB at both Cys82 and Cys139 of CpeA but very inefficiently; the yield of PEB ligated at Cys82 was much lower than observed with CpeY or CpeY/CpeZ. However, CpeS efficiently attached PEB to Cys80 of CpeB but neither CpeY, CpeZ, nor CpeY/CpeZ could ligate PEB to CpeB.

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