Structural studies of Betaine Homocysteine Methyl Transferase (BHMT) and a Dimeric Mutant by Conventional and 2DCOS Moving Lapse IR Spectroscopy

Abstract

Betaine homocysteine S-methyltransferase (BHMT, EC 2.1.1.5) is one of the two enzymes known to methylate homocysteine (Hcy) to generate methionine in the liver. The increase in plasma levels of Hcy (homocysteine) has been established as an independent risk factor for cardiovascular and Alzheimer diseases. BHMT uses betaine as the methyl donor to synthesize methionine, allowing recovery of one of the methyl groups used in choline synthesis by transmethylation. Changes in BHMT activity have been detected under several dietary conditions, during development and in pathologies such as cirrhosis and hepatocellular carcinoma. Rat liver BHMT is a 407-aminoacid cytosolic protein that is more than 90% identical at the amino acid level with its human and pig counterparts. The enzyme contains zinc co-ordinated to three conserved cysteine residues and assembles as a homotetramer. BHMT possesses seven tryptophan residues per subunit located along the sequence including the dimerization arm. Also they are implied in the dimerization arm. We have used a conventional and 2DCOS infrared approach to make a structural study of the differences between BHMT and the dimeric W325F mutant. In these studies, temperature has been used as perturbation. We have also used to extract information from the 2DCOS maps a ‘‘moving lapse’’ approach which uses a narrow frame of 2D spectra along perturbation in order to highlight local variations in structure instead of attending global changes. The mutant has a different amide I shape with a decrease in the area percentages of α-helix and an increase in the band at 1624 cm-1 attributed to extended structures. The moving lapse approach shows the sequence of changes and the structural difference between BHMT and the mutant.