Figure 8

Validation of Thr219–Thr221 as phosphorylation sites and role in homocysteine regulation.

(A) Validation of Thr219–Thr221 as phosphorylation sites in M. smegmatis. SahH-WT and SahH-T219A/T220A/T221A were purified from M. smegmatis, separated by two-dimensional PAGE and immunoblotted with anti-SahH antibodies. For clarity only the region containing SahH isoforms has been shown and different isoforms have been encircled. As seen, one isoform is lost in SahH-T219A/T220A/T221A. (B) Role of Thr219–Thr221 residues in regulating homocysteine concentration. Histogram shows intracellular homocysteine concentration in M. smegmatis MC² 4517 with and without the over-expression of SahH-T219A/T220A/T221A (marked as SahH^tri). Homocysteine concentration was analyzed using UPLC and plotted as μmoles/10¹⁵ CFU. Homocysteine levels were found to be unaffected by SahH-T219A/T220A/T221A (see also Table 1). Error bars represent SD of three independent results.