We first tested the idea of adding a bulky group to the N6
-position of the adenine ring of SAM. To generate necessary extra space, either Leu13 was substituted with Ala, or the C-terminal Asn119 was removed (). Almost no activity was detected after an overnight reaction for both vSET variants in the presence of N6
(; ). Similar results were obtained when N6
-methyl-SAM or N6
-isopropyl-SAM was used (data not shown). Note that the previously reported yeast RMT1-E117G/N6
-benzyl-SAM pair yielded only 0.2% arginine methyltransferase activity as compared to the WT-RMT1/SAM pair.10
Given that the adenine ring forms a network of hydrogen bonds with vSET residues Leu13, His70 and Asn119, our results combined with those from the RMT1-E117G/N6
-benzyl-SAM pair argue that the N6
-position of the adenine is not suitable for generating SAM derivatives that would retain significant methylation activity with a paired vSET variant.
Mass spectrometry data showing the activity of different enzyme/cofactor pairs.
vSET/cofactor pairs with no activity
We next characterized Tyr109 variants with SAM or its analogues. Mass spectrometry analysis shows that Y109G has very little activity in methylating H3K27 with SAM compared to the wild-type vSET (). The same is true with Y109A (data not shown). We then tested the activity of Tyr109 variants in the presence of 16a
. The Y109G/16a
pair was much less active than wild-type/SAM pair as judged by the amount of methylation signal generated in the mass spectrometry study (). However, it was slightly more active than Y109G/SAM and Y109G/16b
pairs (). The activity of Y109A was lower in the presence of SAM analogues than Y109G (data not shown). Therefore, our further studies were carried out with the Y109G variant. To better compare different enzyme/cofactor pairs we performed Michaelis-Menton enzyme kinetic study to obtain the kcat
values (; Supplemental Figure 1
kcat and Km of the vSET/cofactor pairs
When SAM is used, change of Tyr109 to Gly causes a 8,125-fold drop in kcat but little change to Km (). It suggests the substitution at Tyr109 is most likely to directly interfere with its possible function in substrate lysine binding. For Y109G, changing the cofactor from SAM to 16a results in a small increase of kcat but a much higher Km. The same cofactor change for the wild-type vSET causes a 28-fold decrease of catalytic efficiency but surprisingly a 1.5-fold decrease of Km. The increase in Km could be explained by the contribution of fortuitous interaction between the benzyl group and some protein residues. The extremely high Km value for Y109G/16a might be associated with the entropic penalty of fixing the benzyl group at a specific orientation for its insertion into the artificial cavity. Thus, we postulated that a second benzyl group at the 2′-position might restrict the conformation of 3′-benzyl, securing its insertion into the cavity. An additional 2′-benzyl group in SAM (i.e. 2′,3′-dibenzyl-SAM 16c) brings little change of the kcat/Km value to the wild-type vSET with an increase both in kcat and Km. But, a very different scenario was observed for Y109G - a slight increase of kcat but a much-improved Km ().
To mimic the hydrogen bond between Tyr109 and the 3′-hydroxyl of SAM, we synthesized another SAM analogue 19
which bears a carbamoylmethyl substituent at both 2′- and 3′-oxygen atoms, and tested its activity with Y109H, Y109D or Y109N (Supplemental Scheme 1
). We postulated that the amide moiety of the carbamoylmethyl substituent of SAM could form a hydrogen bond with the side chain on the residue replacing Tyr109 in these variants. However, no activity was detected for any of these vSET variants after an overnight incubation, neither was any of these variants active with SAM or the wild-type vSET with 19
. These results emphasize the importance of Tyr109 in substrate binding, and thus change of Tyr109 to His, Asp, or Asn is detrimental to vSET activity.
Inspection of residues around the ribose moiety of SAH in the crystal structure suggests that changing Leu116 could create a docking site for a benzyl group at the 2′- or 3′-position (). Indeed, most of the H3 peptide was converted to mono- or di-methylated species after 19 h of reaction for L116A/16a (). The variant was even more active with 16c as almost all the H3 peptide was converted to tri-methylated species (). Kinetic studies further revealed kcat of 7.4 min−1 and Km of 60 μM for L116A/SAM, which notably is even better than the wild-type vSET/SAM pair (). When 16a was used, the kcat of L116A was dropped to 0.15 min−1, accompanied by a slight increase of Km. Importantly, L116A/16c was more active than WT/16c, with a 3.5-fold higher kcat and a 2.4-fold lower Km. Comparison of the kcat/Km value of L116A/16c with that of L116A/16a argues that the presence of the benzyl groups at both 2′- and 3′-positions have a synergistic effect on L116A catalytic efficiency when compared to the single benzyl group analogues.
Since the presence of the 3′-benzyl in SAM resulted in a reduction in the enzymatic activity of both the wild-type vSET and L116A variant as compared to SAM, we investigated whether 16b would be more potent for L116A. The results show that for the wild-type vSET, the removal of the 3′-benzyl in 16c causes a small decrease in both kcat and Km with a net result of a 1.4-fold decrease in the kcat/Km value. However, for L116A, a 13-fold decrease of kcat was seen with an overall 19-fold decrease in kcat/Km. Interestingly, while adding a single benzyl to the 2′- or 3′-position in SAM causes an ~120-fold decrease in kcat/Km for L116A, these two benzyl groups in 16c likely act synergistically, leading to only a 7-fold decrease of kcat/Km.
Histone lysine methylation is a fundamental mechanism for epigenetic control of gene expression in chromatin. Chemical modulators capable of controlling the enzymatic activity of specific lysine methyltransferases are extremely valuable tools. In this study, we illustrated our approach with an H3K27-specific methyltransferase vSET and SAM. We selected the common N6-position of the adenine ring as well as the novel 2′- and 3′-hydroxyl groups of the ribose as the modification sites. We synthesized seven SAM analogues and characterized their activity with the wild-type and variant vSET proteins. We succeeded in engineering a paired vSET-L116A/16c, which is only slightly less active than vSET-L116A/SAM but more active than the wild-type vSET/16c. Given the presence of SAM in cells, further optimization via chemical modifications of the enzyme and the cofactor is needed to generate a paired vSET variant/modified SAM with activity better than that of the variant with SAM. In summary, our study demonstrates the feasibility of developing a methyl donor cofactor that controls a histone lysine methyltransferase, which could be a useful tool to study the effects of histone H3K27 methylation in vivo.