Published: May 18, 2011 r2011 American Chemical Society 7310 dx.doi.org/10.1021/jf200542w | J. Agric. Food Chem. 2011, 59, 7310–7316 ARTICLE pubs.acs.org/JAFC Biosynthesis of Methoxypyrazines: Elucidating the Structural/ Functional Relationship of Two Vitis vinifera O-Methyltransferases Capable of Catalyzing the Putative Final Step of the Biosynthesis of 3-Alkyl-2-Methoxypyrazine. Jos  e G. Vallarino, †,^ Xaviera A. L opez-Cort  es, ‡,^ Jake D. Dunlevy, §,|| Paul K. Boss, || Fernando D. Gonz  alez-Nilo, ‡ and Yerko M. Moreno †, * † Centro Tecnol ogico de la Vid y el Vino, Facultad de Ciencias Agrarias, Universidad de Talca, Chile ‡ Centro de Bioinform atica y Simulaci on Molecular, Universidad de Talca, Chile § School of Biological Sciences, Flinders University of South Australia, Australia. ) CSIRO Plant Industry, Glen Osmond, Australia ABSTRACT: 3-Alkyl-2-methoxypyrazines (MPs) are an important food constituent and have been associated with detrimental herbaceous flavors in red wines by consumers and the wine industry. The Vitis vinifera genes O-methyltransferase 1 and 2 (VvOMT1 and VvOMT2) have been isolated in the grapevine cultivar Carmenere. These genes encode S-adenosyl-L-methionine (SAM)- dependent O-methyltransferases, which have the ability to methylate 3-alkyl-2-hydroxypyrazines (HPs)—the putative final step in MPs production. Atomic studies were performed in order to explain the differences in these VvOMT activities through their structural/functional relationship in MPs biosynthesis. Differences in enthalpy energy observed between the proteins may be due to changes of equivalent residues in the active sites of VvOMT1 (F319, L322) and VvOMT2 (L319, V322). However, docking simulations and QM/MM analyses described how residues H272 and M182 could explain the main functional differentiation observed between VvOMT1 and VvOMT2 through steric impediment, which limits the formation of the transition state in enzymes encoded by VvOMT2. Therefore, this finding could explain the decreasing catalytic efficiency observed for VvOMT2. KEYWORDS: methoxypyrazines, methyltransferases, Vitis vinifera, wine, flavor, docking simulation, QM/MM ’ INTRODUCTION Nature is full of aromas and flavors, and they are directly linked to food and beverage preference and palatability. Pyrazines are compounds widely distributed in the plant and animal kingdoms and include some of the most potent odorants known, such as the 3-alkyl-2-methoxypyrazines (MPs), which have extremely low sensory detection thresholds as low as 2 ppt in water and 15 ppt in wine. 1 MPs are an important group of natural flavor constituents of some foods and raw vegetables 2À5 including grapes (Vitis vinifera). 6,7 In this regard, 3-isobutyl-2-methoxypyr- azine (IBMP) and 3-isopropyl-2-methoxypyrazine (IPMP) have been found to play an important role in the detrimental herbac- eous flavor of several red wine cultivars such as Cabernet Sauvignon, Merlot, 8 Cabernet Franc, 1 and Carmenere. 9 How- ever, it also contributes to a more acceptable vegetative varietal aroma in white wine cultivars such as Sauvignon blanc, Char- donnay, Semillon, and Riesling. 10,11 The main aromatic descrip- tor associated with IBMP is described as a green pepper aroma, which provides unappealing features in Carmenere red wines. Furthermore, this cultivar produces the highest levels reported in the literature, and it is a recurrent aromatic attribute when describing these wines. 9 The first report on the role of MPs in the flavor of grapes or wine was in 1975 concerning the Cabernet Sauvignon cultivar. 6 Many papers have since been published and report on MPs concentrations in different grapevine tissues, grape berries, and wines of different cultivars and the association of this compound with green or vegetal aromas. 1,3À10,12,13 Different factors can affect the presence of MPs in grapes and wines such as climate and soil, temperature, light, grape ripening, as well as viticultural and enological practices. Climatic conditions have a critical effect upon MPs concentration in grapes. 9 Vines planted in cool climates tend to contain higher levels of MPs, 14 but it is unclear whether this is due to higher MPs production or a lower degradation rate. 11 The role that light/temperature plays in the degradation of MPs is not fully understood. 11,15 It is known that MPs levels in grapevines decrease during grape ripening, and this occurs after veraison. 1,16 The MPs location and concentration in bunches and berries have also been determined. 7,8 It has been reported that field viticultural practices, such as irrigation re- gimes, planting density, and canopy modifications, can modify the MPs levels in grapes. 12,15,17,18 Enological practices during winemaking such as the amount of skin contact 19 and the presence of stem remnants during vinification also affect wine MPs con- centrations. 20 There are also studies covering the effects of the lady beetle (Harmonia axyridis) or yeast strain (Saccharomyces cerevisiae) used in winemaking and their relation to MPs. 21,22 The accumulation and degradation curves of MPs in grapevines and how some factors can modify its concentration during the Received: October 18, 2010 Accepted: May 18, 2011 Revised: May 16, 2011