The esterase from the thermophilic eubacterium Bacillus acidocaldarius: Structural-functional relationship and comparison with the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus

The esterase from the thermophilic eubacterium Bacillus acidocaldarius is a thermophilic and thermostable monomeric protein with a molecular mass of 3 4 KDa, The enzyme, characterized as a "B-type" carboxylesterase, displays t he maximal activity at 65 degrees C, Interestingly, it is also quite active at room temperature, an unusual feature for an enzyme isolated from a ther mophilic microorganism, We investigated the effect of temperature on the st ructural properties of the enzyme, and compared its structural features wit h those of the esterase from the hyperthermophilic archaeon Archaeoglobus f ulgidus. In particular, the secondary structure and the thermal stability o f the esterase were studied by FT-IR spectroscopy, while information on the conformational dynamics of the enzyme were obtained by frequency-domain fl uorometry and anisotropy decays, Our data pointed out that the Bacillus aci docaldarius enzyme possesses a secondary structure rich in alpha-helices as described for the esterase isolated from Archaeoglobus fulgidus, Moreover, infrared spectra indicated a higher accessibility of the solvent ((H2O)-H- 2) to Bacillus acidocaldarius esterase than to Archaeoglobus fulgidus enzym e suggesting, in turn, a less compact structure of the former enzyme. The f luorescence studies showed that the intrinsic tryptophanyl fluorescence of the Bacillus acidocaldarius protein was well represented by the three-expon ential model, and that the temperature affected the protein conformational dynamics, The data suggested an increase in the protein flexibility on incr easing the temperature. Moreover, comparison of Bacillus acidocaldarius est erase with the Archaeoglobus fugidus enzyme fluorescence data indicated a h igher flexibility of the former enzyme at all temperatures tested, supporti ng the infrared data and giving a possible explanation of its unusual relat ive high activity at low temperatures. Proteins 2000; 40:473-481. (C) 2000 Wiley-Liss, Inc.