S-adenosyl-L-homocysteine

Methodology/Principal Findings

Herein we report the expression and purification of Yersinia pestis Dam and the development of a continuous fluorescence based assay for DNA adenine methyltransferase activity that is suitable for determining the kinetic parameters of the enzyme and for high throughput screening against potential Dam inhibitors. The assay utilised a hemimethylated break light oligonucleotide substrate containing a GATC methylation site. When this substrate was fully methylated by Dam, it became a substrate for the restriction enzyme DpnI, resulting in separation of fluorophore (fluorescein) and quencher (dabcyl) and therefore an increase in fluorescence. The assays were monitored in real time using a fluorescence microplate reader in 96 well format and were used for the kinetic characterisation of Yersinia pestis Dam, its substrates and the known Dam inhibitor, S-adenosylhomocysteine. The assay has been validated for high throughput screening, giving a Z-factor of 0.71±0.07 indicating that it is a sensitive assay for the identification of inhibitors.

Introduction

To kinetically characterise Dam and its substrates or inhibitors, it was essential to develop an assay that reported the initial rate of reaction. To do this, we have used a hemimethylated break light oligonucleotide which upon turnover yields the fully methylated product. This is cleaved in situ by the restriction enzyme DpnI, resulting in an increase in fluorescence. Having established a direct relationship between the observed fluorescence increase and the methylation activity of Dam with this hemimethylated substrate, we have used the assay to determine the K M and V max of Y. pestis Dam for S-adenosylmethionine and the oligonucleotide substrate. To demonstrate the convenience of this assay, we have used it to determine the K i of the known Dam inhibitor, S-adenosylhomocysteine with Y. pestis Dam.

Measurement of K i of S-adenosylhomocysteine for Y. pestis Dam

The effect of a known Dam inhibitor, S-adenosylhomocysteine on Y. pestis Dam was investigated using the break light assay. From the data shown in , the K i was calculated to be 6.93±2.01 µM and is lower than the reported value for the E. coli enzyme (K i = 41.6±10.4 µM). These experiments demonstrate the application of the assay to the detailed kinetic analysis of a single inhibitor.

Abstract

Homocysteine (Hcy) has been recognized for the past five decades as a risk factor for atherosclerosis. However, the role of Hcy in the pathological changes associated with atherosclerosis as well as the pathological mechanisms triggered by Hcy accumulation is poorly understood. Due to the reversal of the physiological direction of the reaction catalyzed by S-adenosyl-L-homocysteine hydrolase Hcy accumulation leads to the synthesis of S-adenosyl-L-homocysteine (AdoHcy). AdoHcy is a strong product inhibitor of S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases, and to date more than 50 AdoMet-dependent methyltransferases that methylate a broad spectrum of cellular compounds including nucleic acids, proteins and lipids have been identified. Phospholipid methylation is the major consumer of AdoMet, both in mammals and in yeast. AdoHcy accumulation induced either by Hcy supplementation or due to S-adenosyl-L-homocysteine hydrolase deficiency results in inhibition of phospholipid methylation in yeast. Moreover, yeast cells accumulating AdoHcy also massively accumulate triacylglycerols (TAG). Similarly, Hcy supplementation was shown to lead to increased TAG and sterol synthesis as well as to the induction of the unfolded protein response (UPR) in mammalian cells. In this review a model of deregulation of lipid metabolism in response to accumulation of AdoHcy in Hcy-associated pathology is proposed.

3. AdoHcy-Triggered Deregulation of Lipid Metabolism in Yeast

In yeast, the synthesis of PC from PE by the de novo phospholipid methylation pathway is particularly sensitive to AdoHcy accumulation [, ]. Both inhibition of S-adenosyl-L-homocysteine hydrolase and Hcy supplementation results in AdoHcy accumulation and inhibition of phospholipid methylation in yeast []. However, not only phospholipid methylation, but also a methylation-independent branch of lipid metabolism, namely, TAG synthesis, is affected by AdoHcy accumulation in yeast: yeast cells deficient in AdoHcy catabolism or supplemented with Hcy massively accumulate TAG []. Supporting the causal role of impaired phospholipid methylation in the deregulation of TAG metabolism in response to AdoHcy accumulation, it was found that yeast mutants that are deficient in the enzymatic activities required for methylation of PE to PC, cho2 and opi3, also accumulate TAG []. TAG is known to play an important role in buffering excess fatty acids []. Therefore, TAG accumulation under these conditions suggests accumulation of fatty acids and their redirection from phospholipid to TAG synthesis in methylation deficiency in yeast.

4. Phospholipid Methylation and Homocysteine: Impact on Lipid Metabolism in Mammals

AdoHcy inhibits phosphatidylethanolamine N-methyltransferase in vitro and in vivo also in mammals [, , ]. Similarly as in yeast, deficiency of phospholipid methylation in PEMT−/− knockout mice leads to a rapid decrease of the hepatic PC/PE ratio and accumulation of TAG in the liver, in the absence of choline supplementation []. However, TAG accumulation in the livers of these animals appears to be at least in part due to decreased TAG secretion from hepatocytes [].

4. Phospholipid Methylation and Homocysteine: Impact on Lipid Metabolism in Mammals

Elevated levels of Hcy are as well linked to deregulation of lipid metabolism in mammals. CBS−/− knockout mice exhibit severe hyperhomocysteinemia and accumulate AdoHcy in all tissues tested [, ]. These mutant animals show elevated TAG and nonesterified fatty acid levels in the liver and serum and develop hepatic steatosis [, ]. Another genetic disorder that results in moderately elevated Hcy levels, methylenetetrahydrofolate reductase (MTHFR) deficiency, leads to fatty liver development as well as to neuropathology and aortic lipid deposition in mouse models [, ]. Dietary-induced hyperhomocysteinemia in mice also causes fatty liver, further supporting the role of Hcy in deregulation of lipid metabolism in mammals []. In these mice as well as in the CBS−/− knockout mice lipid accumulates in liver rather than in serum [, ].