Functional analysis of chimeras derived from the Sinorhizobium meliloti and Mesorhizobium loti nodC genes identifies regions controlling chitin oligosaccharide chain length

The rhizobial nodulation gene nodC encodes an N-acetylglucosaminyltransfera se that is responsible for the synthesis of chitin oligosaccharides. These oligosaccharides are precursors for the synthesis of the lipo-chitin oligos accharides that induce cell division and differentiation during the develop ment of nitrogen-fixing root nodules in leguminous plants. The NodC protein s of Mesorhizobium loti and Sinorhizobium meliloti yield chitinpentaose and chitintetraose as their main products, respectively. In order to localize regions in these enzymes that are responsible for this difference in produc t chain length, a set of six chimeric enzymes, comprising different combina tions of regions of the NodC proteins from these two bacteria, was expresse d in Escherichia coli. The oligosaccharides produced were analyzed using th in-layer chromatography. The major conclusion from this work is that a cent ral 91-amino acid segment does not play any obvious role in determining the difference in the chain length of the major product. Furthermore, the char acteristically predominant synthesis of chitintetraose by S. meliloti NodC is mainly dependent on a C-terminal region of maximally 164 amino acids, ex change of only this C-terminal region is sufficient to completely convert t he M. loti chitinpentaose synthase into an S. meliloti-like chitintetraose synthase. The N-terminal region of 170 amino acids also plays a role in res tricting the length of the major product to a tetramer. However, the role o f the C-terminal region is clearly dominant, since exchanging the N-termina l region has no effect on the relative amounts of chitintetraose and -penta ose produced when the C-terminal region of S. meliloti NodC is present. The length of a predicted beta-strand around residue 300 in the C-terminal reg ion of various NodC proteins is the only structural element that seems to b e related to the length of the chitin oligosaccharides produced by these en zymes; the higher the amount of chitintetraose relative to chitinpentaose, the shorter the predicted beta-strand. This element may therefore be import ant for the effect of the C-terminal 164 amino acids on chitin oligosacchar ide chain length.