Figure 1
From: Regulation of homocysteine metabolism by Mycobacterium tuberculosis S-adenosylhomocysteine hydrolase
Metabolic pathways and activity of Mtb-SahH.
(A) Homocysteine metabolism and the role of SahH. SahH (green oval box) catalyzes reversible hydrolysis of SAH to homocysteine and adenosine as shown by bi-directional arrows. Numbers in circles denote the enzymes present in M. tuberculosis H37Rv as follows: 1- SahH (Rv3248c), 2- Cystathionine β-synthase (Rv1077), 3- Cystathionine γ-synthase (Rv1079), 4- Adenosine kinase (Rv2202c), 5- Adenylate kinase (Rv0733), 6- Adenosine deaminase (Rv3313c), 7- Methionine synthase (Rv2124c and Rv1133c), 8- S-adenosylmethionine synthetase (Rv1392) and 9- Methyltransferases, which transfer methyl group (CH3) to substrate molecules. (B) SAH-hydrolytic activity of His6-SahH purified from E. coli. 100 nM of holo-SahH was used with 0–100 μM of SAH. A reaction containing 100 μM SAH in the absence of enzyme (No enz.) served as a control for spontaneous SAH-hydrolysis. OD was measured at 415 nm and plotted as a function of time (minutes). SahH activity increased with increasing concentration of SAH. (C) SAH-synthetic activity of His6-SahH purified from E. coli. Reaction was performed for 2 hours using 4 μM of holo-SahH and TLC image was analyzed by autoradiography (lane 1). SahH from Rabbit erythrocytes was used as a positive control (lane 3) and a negative control was setup without the (lane 2). Adenosine (Ado) and SAH spots are indicated by arrows. After the densitometric analysis of spots, percent SAH synthesis is plotted in lower panel. An extra spot above SAH-spot in lane 3 could be inosine due to slight contamination (<1%) of adenosine deaminase enzyme in SahH (from rabbit erythrocytes purchased from a commercial source, Sigma) that deaminates adenosine to inosine. Corresponding retention factors (Rf) were SAH (0.1), inosine (0.36) and adenosine (0.5).
