Structural insights into cold inactivation of tryptophanase and cold adaptation of subtilisin S41

Abstract

A wide variety of enzymes can undergo a reversible loss of activity at low temperature, a process that is termed cold inactivation. This phenomenon is found in oligomeric enzymes such as tryptophanase (Trpase) and other pyridoxal phosphate dependent enzymes. On the other hand, cold‐adapted, or psychrophilic enzymes, isolated from organisms able to thrive in permanently cold environments, have optimal activity at low temperature, which is associated with low thermal stability. Since cold inactivation may be considered “contradictory” to cold adaptation, we have looked into the amino acid sequences and the crystal structures of two families of enzymes, subtilisin and tryptophanase. Two cold adapted subtilisins, S41 and subtilisin‐like protease from Vibrio, were compared to a mesophilic and a thermophilic subtilisins, as well as to four PLP‐dependent enzymes in order to understand the specific surface residues, specific interactions, or any other molecular features that may be responsible for the differences in their tolerance to cold temperatures. The comparison between the psychrophilic and the mesophilic subtilisins revealed that the cold adapted subtilisins have a high content of acidic residues mainly found on their surface, making it charged. The analysis of the Trpases showed that they have a high content of hydrophobic residues on their surface. Thus, we suggest that the negatively charged residues on the surface of the subtilisins may be responsible for their cold adaptation, whereas the hydrophobic residues on the surface of monomeric Trpase molecules are responsible for the tetrameric assembly, and may account for their cold inactivation and dissociation. © 2007 Wiley Periodicals, Inc. Biopolymers 89: 354–359, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Biochemical characterization of an alcohol dehydrogenase from Ralstonia sp.

“…The apparently lower stability at 0°C could be a consequence of cold inactivation. This phenomenon which has been observed with several oligomeric enzymes is assumed to be a consequence of weakened hydrophobic interactions at subunit interfaces (Almog et al, 2007; Erez et al, 2002; Ishikura et al, 2003). RADH shows highest stability at 15°C (half‐life: 138 h, Fig.…”

New tryptophanase inhibitors: Towards prevention of bacterial biofilm formation

“…In addition, it requires certain monovalent cations (K þ , NH þ 4 , TI þ ) for activity and for tight PLP binding [4 -6]. The enzyme undergoes a reversible inactivation followed by dissociation into dimers or monomers, depending on the bacteria species, after incubation for several hours at 28C [7]. The enzymatic reaction with tryptophan is shown in Scheme 1.…”

Cold Adapted Subtilases

“…It is postulated that optimization of the catalytic properties is achieved through increased local or global molecular flexibility, facilitating the conformational changes required for catalytic function at low temperatures [60À63]. None of these structural features is present in all cold adapted enzymes, however, and results show that each protein family adopts its own strategies in coping with temperature adaptation [3,13,63,66]. Decreased structural stability in comparison to related homologous enzymes from meso-and thermophilic organisms is indeed a characteristic feature of cold adapted enzymes [60,63À65].…”

“…Decreased structural stability in comparison to related homologous enzymes from meso-and thermophilic organisms is indeed a characteristic feature of cold adapted enzymes [60,63À65]. Based on structural analysis, a common feature of the cold adapted subtilases is the anionic character of their molecular surfaces, arising from uncompensated negative charges of Asp and Glu residues [10,13,14,66]. Several comparative studies of metagenomic and genomic data from psychrophilic organisms have been in agreement with these results, but have also indicated that cold enzymes contain relatively high levels of polar uncharged amino acid residues and significant trends for amino acid substitutions leading to higher structural flexibility (for review see: Casanueva et al [3]).…”

“…Among the cold adapted subtilases, some of these structural features, e.g. While the different calcium binding sites are shown to be crucial for both kinetic and thermodynamic stabilization of the subtilases [48], differences in the multiple calcium binding sites do not seem to be structural determinants in their cold adaptation [66]. Based on structural analysis, a common feature of the cold adapted subtilases is the anionic character of their molecular surfaces, arising from uncompensated negative charges of Asp and Glu residues [10,13,14,66].…”