6.3. Identification of the amino acids important for the glycosyltransferase activities associated with human LH3 and C. elegans LH (III, IV)

6.3.1. Active sites of GGT in human LH3 and C. elegans LH (III)

The data from site-directed mutagenesis combined with in vitro translation and E. coli expression system revealed that the active sites of GGT are located in the amino-terminal parts of the molecule in C. elegans LH and human LH3. There are evolutionary changes between C. elegans LH and human LH3, the active sites are not identical in these species although GT and GGT activities are present in both molecules. C. elegans LH seems to be more sensitive to the LH3-C. elegans specific amino acid mutations, as more inhibitory changes were found at the amino-terminal part of the molecule for C. elegans LH, and the inhibitions were more effective compared with that of human LH3. Two mutations, the Cys₁₄₄Ile and Leu₂₀₈Ile in human LH3 sequence, decreased GGT activity dramatically but had no effect on LH activity. The shortening of the carboxy-terminal portion of LH3 eliminates LH activity, but only reduces GGT activity. These findings provide further evidence that LH and GGT active sites are separated on the LH3 molecule. This is consistent with the earlier data obtained from LH1 that the amino acids responsible for LH activity are located in the carboxy-terminal portion of the molecule (Pirskanen et al. 1996, Passoja et al. 1998a), the most conserved region among LH isoforms (Hautala et al. 1992, Valtavaara et al. 1997, Passoja et al. 1998b, Valtavaara et al. 1998, Ruotsalainen et al. 1999). This result is also confirmed by a recent study in which LH3 was produced as a secreted form in baculovirus system (Rautavuomo et al. 2002). Our data indicated that the Cys₁₄₄ is not disulfide linked whereas the Cys₄₉₄ and the Cys₅₇₇ used as controls are disulfide bonded. Furthermore our data suggest that although the Cys₁₄₄ is required for the functional integrity of GGT, it probably does not participate directly in the enzyme catalysis.

Sequence alignments and X-ray crystal structures have indicated a so-called DxD motif in many glycosyltransferases, and it has been found in different glycosyltransferase families (Ünligil et al. 2000). This motif is thought to stabilize the Mn2+ and thus indirectly stabilize the binding of the diphosphate moiety of the UDP-sugar (Ünligil et al. 2000, Gastinel et al. 2001). At least three DxD motifs exist in the LH3 molecule. Only the mutations of the most amino-terminal DxD motif, which consists of five aspartates at position 187-191, eliminated GGT activity, indicating that this region is important for GGT activity and might act as a Mn2+ binding site in the molecule. However, this needs to be verified by crystallization of LH3 protein.

The replacement of the two most conserved amino acids among the LH3-C. elegans specific amino acids, the Cys₁₄₄ and Leu₂₀₈, in the LH1 and LH2 structures is not sufficient to restore GGT activity to these isoforms, suggesting that LH1 and LH2 have diverged so much during evolution from the ancestral LH3 (Ruotsalainen et al. 1999) that single amino acid changes are not sufficient to restore glycosyltransferase activity.

6.3.2. Active sites of GT in human LH3 (IV)

The search for GT active sites was carried out by in vitro mutagenesis combined with protein expression in E. coli BL21(DE3)pLysS cells with the pET-15b vector. The data indicate that the mutations of Cys₁₄₄Ile, and aspartates at position 187-191 reduced the GT activity remarkably whereas the mutations at the carboxy-terminal end of the molecule and the DxD motif at position 392-394 had no effect on GT activity. The results thus suggest that the amino acids located at the amino-terminal part of the molecule, important for GGT activity, are also required for GT activity. The data indicate similar results for GT and GGT. More in vitro mutagenesis studies are needed to see whether GT active site is identical to that of GGT.