Structures of two zinc -dependent homocysteine S -methyltransferases: Betaine -homocysteine methyltransferase and cobalamin -dependent methionine synthase.

Homocysteine (Hcy) has been identified as an independent risk factor for a variety of health problems that includes cardiovascular disease, neural tube defects and Alzheimer's disease. Betaine-homocysteine methyltransferase (BHMT) and cobalamin-dependent methionine synthase (Metes) are the two mammalian enzymes that convert homocysteine to methionine. BHMT and Metes are evolutionarily related and share a common mechanistic strategy, using a catalytic zinc ion to bind and activate homocysteine. Structures for these proteins and their roles in enzyme chemistry have not until now been described. The structures of human BHMT and a construct comprising N-terminal substrate-binding domains from Thermotoga maritima Metes were solved using X-ray crystallographic methods. Crystals of these constructs were obtained from extensive screening and optimization. Experimental phase information was calculated using single and multiple wavelength anomalous diffraction (SAD/MAD), using a samarium derivative of BHMT and a selenomethionyl derivative of T. maritima Metes. Substrate complexes of the two enzymes were determined using molecular replacement. The Hcy binding domains from BHMT and Metes are both (beta/alpha) 8 barrels containing significant distortions from classical geometry to assemble the zinc binding site; strands beta6 and beta8 are drawn together with strand beta7 diverted, and strands beta1 and beta2 are spread apart. BHMT and MetH have evolved different binding sites their respective methyl donors, glycine betaine and methylcobalamin. BHMT forms a hydrophobic ring around betaine, whereas the binding site for methylcobalamin in MetH is a deep socket that accommodates the large corrin macrocycle. A low dielectric environment surrounds Hcy in both enzymes. The Hcy domain of MetH is part of a multidomain protein, and the structure indicates the two domains are packed together in a rigid structural unit with fixed orientations. The cobalamin cofactor must travel a distance of >50 A in order to accept a methyl group from methyl tetrahydrofolate and carry it to Hcy. Fixing the domains together increases the efficiency of primary turnover by simplifying the search for substrates.