No abstract

Chronic inflammation activates the tryptophan-degrading enzyme IDO, which is well known to impair T cell proliferation. We have previously established that bacille Calmette-Guérin (BCG), an attenuated form of Mycobacterium bovis, is associated with persistent activation of IDO in the brain and chronic depressive-like behavior, but a causative role has not been established. In these experiments we used both pharmacologic and genetic approaches to test the hypothesis that IDO activation is responsible for the development of chronic depression that follows BCG infection. BCG induced TNF-α, IFN-γ, and IDO mRNA steady-state transcripts in the brain as well as the enzyme 3-hydroxyanthranilic acid oxygenase (3-HAO) that lies downstream of IDO and generates the neuroactive metabolite, quinolinic acid. Behaviors characteristic of depression were apparent 1 wk after BCG infection. Pretreatment with the competitive IDO inhibitor 1-methyltryptophan fully blocked BCG-induced depressive-like behaviors. Importantly, IDO-deficient mice were completely resistant to BCG-induced depressive-like behavior but responded normally to BCG induction of proinflammatory cytokines. These results are the first to prove that the BCG-induced persistent activation of IDO is accompanied by the induction of 3-hydroxyanthranilic acid oxygenase and that IDO is required as an initial step for the subsequent development of chronic depressive-like behavior.

We demonstrate that purified recombinant human betainehomocysteine methyltransferase-2 (BHMT-2) is a zinc metalloenzyme that uses S-methylmethionine (SMM) as a methyl donor for the methylation of homocysteine. Unlike the highly homologous betaine-homocysteine methyltransferase (BHMT), BHMT-2 cannot use betaine. The K m of BHMT-2 for SMM was determined to be 0.94 mM, and it has a turnover number similar to BHMT. Several compounds were tested as inhibitors of recombinant human BHMT and BHMT-2. The SMM-specific methyltransferase activity of BHMT-2 is not inhibited by dimethylglycine and betaine, whereas the former is a potent inhibitor of BHMT. Methionine is a stronger inhibitor of BHMT-2 than BHMT, and S-adenosylmethionine does not inhibit BHMT but is a weak inhibitor of BHMT-2. BHMT can use SMM as a methyl donor with a k cat /K m that is 5-fold lower than the k cat /K m for betaine. However, SMM does not inhibit BHMT activity when it is presented to the enzyme at concentrations that are 10-fold greater than the subsaturating amounts of betaine used in the assay. Based on these data, it is our current hypothesis that in vivo most if not all of the SMM-dependent methylation of homocysteine occurs via BHMT-2.Homocysteine (Hcy) 3 is derived from methionine (Met) and can either be methylated to reform Met (i.e. remethylation) or participate in cysteine biosynthesis via the transsulfuration pathway. Hcy remethylation in mammals has always been attributed to two different enzymes: cobalamin-dependent methionine synthase and betaine-homocysteine methyltransferase (BHMT; EC 2.1.1.5). Methionine synthase uses 5-methyltetrahydrofolate as the methyl donor and is expressed in all tissues at very low levels, whereas BHMT uses betaine (Bet) as the methyl donor and is only expressed in the liver and kidney, but at very high levels (1-3).Apart from the mammalian methyltransferases described above, the existence of other Hcy methyltransferase (HMT) activities in rat liver extracts, namely S-methylmethionine (SMM)-and S-adenosylmethionine (AdoMet)-HMT, were reported by Shapiro and Yphantis in the late fifties (4). SMM, also known as vitamin U, is an analog of AdoMet, with a methyl group substituted for the adenosyl group. This amino acid is a major sulfur containing metabolite of many plants (5-7) but it is not known to be synthesized by mammals.In 2000, Chadwick et al. (8) reported mouse and human cDNA sequences whose deduced amino acid sequences had very high homology to BHMT. They were named Bhmt-2 and BHMT-2, respectively, even though their enzymatic activities were not determined. Like human BHMT, the human BHMT-2 mRNA was shown to be abundantly expressed in liver and kidney. BHMT and BHMT-2 are adjacent to each other on human chromosome 5 (5q13), suggesting they are tandem duplicates. We demonstrate herein that the translational product of the cDNA named BHMT-2 is a zinc metalloenzyme that methylates Hcy using SMM, and to a much lesser extent, AdoMet as methyl donors in vitro. We also show that purified recombinant human BHM...

status modulates the induction of hepatic glycine N-methyltransferase and homocysteine metabolism in diabetic rats. Am J Physiol Endocrinol Metab 291: E1235-E1242, 2006. First published July 11, 2006 doi:10.1152/ajpendo.00237.2006.-A diabetic state induces the activity and abundance of glycine N-methyltransferase (GNMT), a key protein in the regulation of folate, methyl group, and homocysteine metabolism. Because the folate-dependent one-carbon pool is a source of methyl groups and 5-methyltetrahydrofolate allosterically inhibits GNMT, the aim of this study was to determine whether folate status has an impact on the interaction between diabetes and methyl group metabolism. Rats were fed a diet containing deficient (0 ppm), adequate (2 ppm), or supplemental (8 ppm) folate for 30 days, after which diabetes was initiated in one-half of the rats by streptozotocin treatment. The activities of GNMT, phosphatidylethanolamine Nmethyltransferase (PEMT), and betaine-homocysteine S-methyltransferase (BHMT) were increased about twofold in diabetic rat liver; folate deficiency resulted in the greatest elevation in GNMT activity. The abundance of GNMT protein and mRNA, as well as BHMT mRNA, was also elevated in diabetic rats. The marked hyperhomocysteinemia in folate-deficient rats was attenuated by streptozotocin, likely due in part to increased BHMT expression. These results indicate that a diabetic state profoundly modulates methyl group, choline, and homocysteine metabolism, and folate status may play a role in the extent of these alterations. Moreover, the upregulation of BHMT and PEMT may indicate an increased choline requirement in the diabetic rat.choline; phosphatidylethanolamine; betaine-homocysteine S-methyltransferase THE FOLATE-DEPENDENT ONE-CARBON POOL and methyl group metabolism are interrelated pathways that are critically important in optimal health, as perturbation of these metabolic processes is associated with a number of pathologies, including cardiovascular disease, cancer development, and birth defects ( Fig. 1) (26,43,46). The primary methyl group donor, S-adenosylmethionine (SAM), requires a constant supply of methyl groups from the diet and/or the one-carbon pool for numerous transmethylation reactions, such as the synthesis of phosphatidylcholine (PC) by the action of the liver-specific enzyme phosphatidylethanolamine N-methyltransferase (PEMT) (31). Therefore, it is essential to regulate the supply and utilization of methyl groups to optimize SAM-dependent transmethylation reactions, a function that is accomplished by the enzymatic activity of a key regulatory protein, glycine N-methyltransferase (GNMT). GNMT is an abundant protein in the liver, comprising ϳ1-3% of all hepatic cytosolic protein, and has also been identified in renal and pancreatic tissue (36, 61). GNMT optimizes the SAM/S-adenosylhomocysteine (SAH) ratio by catalyzing the conversion of SAM and glycine to SAH and sarcosine, respectively (5, 17). Because SAH is a potent inhibitor of methyltransferase activity (28), optimizing the SA...

RSR:I [N:6-adenine] DNA methyltransferase (M.RSR:I), which recognizes GAATTC and is a member of a restriction-modification system in Rhodobacter sphaeroides, was purified to >95% homogeneity using a simplified procedure involving two ion exchange chromatographic steps. Electrophoretic gel retardation assays with purified M.RSR:I were performed on unmethylated, hemimethylated, dimethylated or non-specific target DNA duplexes (25 bp) in the presence of sinefungin, a potent inhibitory analog of AdoMet. M. RSR:I binding was affected by the methylation status of the DNA substrate and was enhanced by the presence of the cofactor analog. M. RSR:I bound DNA substrates in the presence of sinefungin with decreasing affinities: hemimethylated > unmethylated > dimethylated >> non-specific DNA. Gel retardation studies with DNA substrates containing an abasic site substituted for the target adenine DNA provided evidence consistent with M.RSR:I extruding the target base from the duplex. Consistent with such base flipping, an approximately 1.7-fold fluorescence intensity increase was observed upon stoichiometric addition of M.RSR:I to hemimethylated DNA containing the fluorescent analog 2-aminopurine in place of the target adenine. Pre-steady-state kinetic and isotope- partitioning experiments revealed that the enzyme displays burst kinetics, confirmed the catalytic competence of the M.RSR:I-AdoMet complex and eliminated the possibility of an ordered mechanism where DNA is required to bind first. The equilibrium dissociation constants for AdoMet, AdoHcy and sinefungin were determined using an intrinsic tryptophan fluorescence-quenching assay.