Crystal structure of 3-isopropylmalate dehydrogenase from the moderate facultative thermophile, Bacillus coagulans: two strategies for thermostabilization of protein structures.

Abstract

The crystal structure of 3-isopropylmalate dehydrogenase from the moderate facultative thermophile Bacillus coagulans (BcIPMDH) has been determined by the X-ray method. BcIPMDH is a dimeric enzyme composed of two identical subunits, each of which takes an open alpha/beta structure with 11 alpha-helices and 14 beta-strands. The polypeptide is folded into two domains. The first domain is composed of residues 1-101 and 257-356, and the second domain, of residues 102-256. The latter domains of the two subunits are associated with one another by a dyad axis to make the dimer, locally forming a beta-sheet and a four-helix bundle. As compared with the structure of the enzyme from the extreme thermophile Thermus thermophilus (TtIPMDH), a new short beta-sheet (residues 329-330 and 340-341) absent in TtIPMDH is formed by the insertion of 5 residues in BcIPMDH. In terms of determinants for thermostabilization, both consistent and inconsistent changes were found between the two enzymes. The regions including inconsistent changes are formed by different usages of the determinants for stabilizing the loops at different levels. Those in BcIPMDH contain some structural redundancies in length of amino acid sequence and flexibility of residues, which seem to be unnecessary for the enzymatic reaction. Such redundancies are also found in the primary structure of the enzyme of the mesophile Bacillus subtilis, but these parts are more stabilized in BcIPMDH by hydrogen bonds and salt bridges. On the other hand, TtIPMDH is stabilized by reducing such redundant parts. This contrast suggests that different strategies may be preferred for thermostabilization, depending on temperature.