Abstract
Thermotoga maritima invertase (-fructosidase) hy-drolyzes sucrose to release fructose and glucose, which are major carbon and energy sources for both pro-karyotes and eukaryotes. The name " invertase " was given to this enzyme over a century ago, because the 1:1 mixture of glucose and fructose that it produces was named " invert sugar. " Despite its name, the enzyme operates with a mechanism leading to the retention of the anomeric configuration at the site of cleavage. The enzyme belongs to family GH32 of the sequence-based classification of glycosidases. The crystal structure, determined at 2-Å resolution, reveals two modules, namely a five-bladed-propeller with structural similarity to the-propeller structures of glycosidase from families GH43 and GH68 connected to a-sandwich module. Three carboxylates at the bottom of a deep, negatively charged funnel-shaped depression of the-propeller are essential for catalysis and function as nucleophile, general acid, and transition state stabilizer, respectively. The catalytic machinery of invertase is perfectly super-imposable to that of the enzymes of families GH43 and GH68. The variation in the position of the furanose ring at the site of cleavage explains the different mechanisms evident in families GH32 and GH68 (retaining) and GH43 (inverting) furanosidases. Invertase, the-D-fructofuranosidase (EC 3.2.1.26) that cleaves sucrose into fructose and glucose is one of the earliest discovered enzymes. It was isolated in the second half of the 19th century, and its name was coined because the enzyme produces " invert " sugar, which is a 1:1 mixture of dextrorota-tory D-glucose and levorotatory D-fructose (1). Because of its chemical structure, sucrose can be cleaved by either-glucosi-dase or-fructofuranosidase activity. Koshland and Stein established that invertase is a-fructofuranosidase by performing the reaction in 18 O-labeled water and determining the 18