The reaction was followed by acquiring 1D NMR experiments at 15-min intervals over 24 h. Gene functions were inferred from BLAST searches followed by gene linkage and cluster analysis. Deletion of yjhC resulted in loss of growth on 2,7-anhydro-Neu5Ac but not on Neu5Ac (Fig. 5C), which could be complemented in trans with yjhC (Fig. 5D), suggesting that the gene encodes an equivalent protein to RgNanOx. To test this hypothesis, the YjhC protein was recombinantly expressed and purified, and its activity against 2,7-anhydro-Neu5Ac and Neu5Ac was analyzed by ESI-MS. A, ESI-MS analysis of the enzymatic reaction between RgNanOx mutants and 2,7-anhydro-Neu5Ac (290; left) or Neu5Ac (308; left). A, ESI-MS analysis of the enzymatic reaction of RgNanOx, EcNanOx, and HhNanOx with 2,7-anhydro-Neu5Ac (left) or Neu5Ac (right). Having demonstrated that NanOx-like genes are functional in both Gram-positive and Gram-negative bacteria, functioning with different classes of transporters, we extended our analysis to likely 2,7-anhydro-Neu5Ac catabolic genes across bacterial species. The genes encoding 2,7-anhydro-Neu5Ac transporters, 2,7-anhydro-Neu5Ac oxidoreductases, and IT-sialidases are distinguished by color for emphasis.
TIGR4 possesses both the conserved sialic acid “supercluster,” as in strain D39, and an additional, candidate 2,7-anhydro-Neu5Ac cluster bearing the siaT-like transporter gene. The first gene in the yjhBC operon, yjhB, encodes a major facilitator superfamily (MFS) transporter protein that shows homology (35% identify, 55% similarity) to NanT, the known Neu5Ac transporter in E. coli (24, 26, 27). Deletion of nanT leads to a complete loss of growth on Neu5Ac, suggesting that YjhB cannot transport this particular sialic acid (28) (Fig. 7A). Similar to the phenotype observed with the ΔyjhC strain, the ΔyjhB mutant was also unable to grow on 2,7-anhydro-Neu5Ac but could grow on Neu5Ac (Fig. 7B). The co-expression of these two genes and the requirement of YjhB for growth on 2,7-anhydro-Neu5Ac suggest that YjhB is a novel MFS transporter for 2,7-anhydro-Neu5Ac and that these two genes together form an “accessory” operon to allow E. coli to scavenge a wider range of sialic acids that are available in the human gut. The first locus is the core nanATEKyhcH operon for Neu5Ac uptake and dissimilation into the cytoplasm (62). The two “accessory” loci contain the nanCMS operon, for Neu5Ac uptake through the outer membrane, sialic acid mutarotation, and processing of O-acetylated sialic acids in periplasm (63,-65), and the nanXY (yjhBC) operon here characterized as being required for 2,7-anhydro-Neu5Ac uptake and utilization.
We propose to rename these genes nanXY, because the function of the final NanR-regulated operon has been elucidated through this work. Varki, Glycobiology 2: 25-40 (1992); Sialic Acids: Chemistry, Metabolism and Function , R. Schauer, Ed. A, Neu5Ac lyase; nanK, N-acetylmannosamine kinase; nanE, N-acetylmannosamine-6-phosphate epimerase; nanC, Neu5Ac outer membrane channel; nanM, Neu5Ac mutarotase; nanS, N-acetyl-9-O-acetylneuraminate esterase; nagB, glucosamine-6-phosphate deaminase; GNAT, GCN5-related N-acetyltransferase; Reg, regulator (please note that GNAT family proteins and regulator proteins, while recurrent within clusters, may belong to different clades and thus function differently in each organism); SAT2, 2,7-anhydro-Neu5Ac transporter of the ABC superfamily; siaPQM, Neu5Ac transporter of the TRAP family; satABCD, Neu5Ac transporter of the ABC superfamily (SAT); nanUVW (SAT3), Neu5Ac transporter of the ABC superfamily (also named satABC); nanT, Neu5Ac transporter of the MFS superfamily; siaT, Neu5Ac transporter of the SSS family; nanX (yjhB), 2,7-anhydro-Neu5Ac transporter (nanT-like) of the MFS superfamily ABC; MFS, major facilitator superfamily; SSS, sodium solute symporter family; GPH, glycoside-pentoside-hexuronide:cation symporter family; SBP, solute-binding protein; TMD, transmembrane domain; NBD, nucleotide-binding domain.
Both mutants were grown on 2,7-anhydro-Neu5Ac (orange), Neu5Ac (blue), glucose (red), or M9 medium alone (black) in 200-µl microtiter plates. The red line marks the trajectory of hydride transfer. All strains were grown on 2,7-anhydro-Neu5Ac (orange), Neu5Ac (blue), glucose (red), or M9 medium alone (black) in 200-µl microtiter plates. We found that this can be advantageously done by disrupting the nanA and nanK genes in the strains which will be used for Neu5Ac production. In case you loved this short article and you would love to receive much more information relating to sialic acid powder factory please visit our web site. E. coli K12 the nanA nanK and nanT genes are clustered in the same region of the E. coli chromosome together with the nanE gene which encodes the ManNac kinase activity. NanK NanE NagA GlmM and GlmU catalyse the formation of UDP-GlcNAc from ManNAc. In RgNanOx, by creating a keto group, the enzyme has acidified the C3 proton; this facilitates an elimination reaction and formation of a conjugated intermediate 4-keto-DANA, which we detected by NMR. In other enzymes, including RmlB of the dTDP-l-rhamnose biosynthetic pathway (31) and the multistep enzyme GDP-mannose 3,5-epimerase (32), the creation of a keto group and the consequent acidification of the α proton(s) allow a range of chemical reactions. The creation of a keto intermediate in sugars is widespread in biology; perhaps it is best-known for the SDR enzyme UDP-glucose/galactose epimerase (29, 30). In this enzyme, the oxidation and reduction of the sugar occur so as to invert the chirality at C4.