The lipopolysaccharide (LPS) of the deep rough mutant Haemophilus influenzae I69 consists of lipid A and a single 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residue substituted with one phosphate at position 4 or 5 (Helander, I. M., Lindner, B., Brade, H., Altmann, K., Lindberg, A. A., Rietschel, E. T., and Zähringer, U. (1988) Eur. J. Biochem. 177, 483-492). The waaA gene encoding the essential LPS-specific Kdo transferase was cloned from this strain, and its nucleotide sequence was identical to H. influenzae DSM11121. The gene was expressed in the Gram-positive host Corynebacterium glutamicum and characterized in vitro to encode a monofunctional Kdo transferase. waaA of H. influenzae could not complement a knockout mutation in the corresponding gene of an Re-type Escherichia coli strain. However, complementation was possible by coexpressing the recombinant waaA together with the LPS-specific Kdo kinase gene (kdkA) of H. influenzae DSM11121 or I69, respectively. The sequences of both kdkA genes were determined and differed in 25 nucleotides, giving rise to six amino acid exchanges between the deduced proteins. Both E. coli strains which expressed waaA and kdkA from H. influenzae synthesized an LPS containing a single Kdo residue that was exclusively phosphorylated at position 4. The structure was determined by nuclear magnetic resonance spectroscopy of deacylated LPS. Therefore, the reaction products of both cloned Kdo kinases represent only one of the two chemical structures synthesized by H. influenzae I69.

transfer, Haemophilus influenzae, Escherichia coli, 3-deoxy-D-manno-oct-2-ulosonic acid, Kdo, transferase, lipid A, Legionella pneumophila, mutation

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8
0,6,6,4	P
0,6,6	aXKdop	172.6	99.3	33.2	70.8	65.4	72.0	69.2	63.2
0,6,4	P
0,6	bDGlcpN	99.2	55.8	71.9	74.4	74.0	?
0	aDGlcpN	92.0	54.2	69.6	69.7	72.8	69.2
	P


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8
0,6,6,4	P
0,6,6	aXKdop	-	-	2.028-2.204	4.535	4.213	3.804	3.948	3.653-3.906
0,6,4	P
0,6	bDGlcpN	4.854	3.127	3.883	3.923	3.746	3.596-3.773
0	aDGlcpN	5.722	3.421	3.898	3.499	4.095	3.861-4.251
	P


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8
0,6,6,4	P
0,6,6	aXKdop			33.2/2.028-2.204	70.8/4.535	65.4/4.213	72.0/3.804	69.2/3.948	63.2/3.653-3.906
0,6,4	P
0,6	bDGlcpN	99.2/4.854	55.8/3.127	71.9/3.883	74.4/3.923	74.0/3.746	?/3.596-3.773
0	aDGlcpN	92.0/5.722	54.2/3.421	69.6/3.898	69.7/3.499	72.8/4.095	69.2/3.861-4.251
	P

The spectrum also has 1 signal at unknown position (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

N[C@H]1[C@@H](OP(=O)(O)O)O[C@H](CO[C@@H]2O[C@H](CO[C@]3(C(=O)O)C[C@@H](OP(=O)(O)O)[C@@H](O)[C@@H]([C@H](O)CO)O3)[C@@H](OP(=O)(O)O)[C@H](O)[C@H]2N)[C@@H](O)[C@@H]1O

800.443 g/mol (C₂₀H₃₉N₂O₂₅P₃)