Bivalent cations and amino‐acid composition contribute to the thermostability of Bacillus licheniformis xylose isomerase

Abstract

Comparative analysis of genome sequence data from mesophilic and hyperthermophilic micro-organisms has revealed a strong bias against specific thermolabile aminoacid residues (i.e. N and Q) in hyperthermophilic proteins. The N þ Q content of class II xylose isomerases (XIs) from mesophiles, moderate thermophiles, and hyperthermophiles was examined. It was found to correlate inversely with the growth temperature of the source organism in all cases examined, except for the previously uncharacterized XI from Bacillus licheniformis DSM13 (BLXI), which had an N þ Q content comparable to that of homologs from much more thermophilic sources. To determine whether BLXI behaves as a thermostable enzyme, it was expressed in Escherichia coli, and the thermostability and activity properties of the recombinant enzyme were studied. Indeed, it was optimally active at 70-72 8C, which is significantly higher than the optimal growth temperature (37 8C) of B. licheniformis. The kinetic properties of BLXI, determined at 60 8C with glucose and xylose as substrates, were comparable to those of other class II XIs. The stability of BLXI was dependent on the metallic cation present in its two metal-binding sites. The enzyme thermostability increased in the order apoenzyme , Mg 2þ -enzyme , Co 2þ -enzyme < Mn 2þ -enzyme, with melting temperatures of 50.3 8C, 53.3 8C, 73.4 8C, and 73.6 8C. BLXI inactivation was first-order in all conditions examined. The energy of activation for irreversible inactivation was also strongly influenced by the metal present, ranging from 342 kJ·mol 21 (apoenzyme) to 604 kJ·mol 21 (Mg 2þ -enzyme) to 1166 kJ·mol 21 (Co 2þ -enzyme). These results suggest that the first irreversible event in BLXI unfolding is the release of one or both of its metals from the active site. Although N þ Q content was an indicator of thermostability for class II XIs, this pattern may not hold for other sets of homologous enzymes. In fact, the extremely thermostable a-amylase from B. licheniformis was found to have an average N þ Q content compared with homologous enzymes from a variety of mesophilic and thermophilic sources. Thus, it would appear that protein thermostability is a function of more complex molecular determinants than amino-acid content alone.Keywords: Bacillus licheniformis; metal binding; thermostability; xylose isomerase.It has become apparent that protein thermostability arises not from a single chemical or physical factor, but from numerous subtle contributions integrated over the entire molecular structure [1 -6]. Thermostable proteins usually exhibit no significant differences in backbone conformation when compared with less thermostable proteins, but they typically have increased numbers of salt bridges, side chain-side chain hydrogen bonds, and residues involved in a helices [7 -9]. Stability at very high temperatures further requires that a particular enzyme resist thermally induced deleterious chemical reactions, which usually occur at insignificant rates at lower temperatures [10]. For example, one of the mos...

Genomic Correlates of Hyperthermostability, an Update

“…This includes strongly reduced frequencies for the thermolabile amino acid residues Gln and Asn, as also noted by Vieille et al (6). Among other trends, the aliphatic residue Val appears to be preferred in hyperthermophiles, whereas the tiny non-polar non-charged residue Ala is avoided.…”

Discrimination of thermophilic and mesophilic proteins

“…Since then, a great many papers have been written on the subject. Some of the proposed mechanisms/indicators of increased thermostability include: a more highly hydrophobic core [7,8], tighter packing or compactness [9], deleted or shortened loops [10,11], greater rigidity [3,12,13] (for example through increased Proline content in loops), higher secondary structure content [14], greater polar surface area [15], fewer and/or smaller voids [14,16], smaller surface area to volume ratio [17], fewer thermolabile residues [16,18], increased hydrogen bonding [15], higher isoelectric point [19], and more salt bridges/ion pairs and networks of salt bridges [6,[20][21][22][23][24][25].…”

Cloning, Overexpression, and Characterization of a Novel Thermostable Penicillin G Acylase from Achromobacter xylosoxidans : Probing the Molecular Basis for Its High Thermostability

“…Early studies indicated that there is a correlation between protein stability and the number of arginines on the protein surface (44). Thermophilic proteins have, on average, a higher arginine content and a greater amount of reducing lysines on the protein surface (1,48). The site-directed mutagenesis study has proved that LysArg mutations increases the thermotolerance of enzymes (6,29,44) because of stronger hydrogen bonding of the large guanidinium group of arginine with nearby polar groups (44).…”

“…The results showed that the PGA650 protein has decreased amounts of amino acid residues of N plus Q and of S plus T (Table 2), which is consistent with the conclusion of the genomic studies conducted by Chakravarty and Varadarajan (5) and Sterner and Liebl (40). They found that mesophilic proteins normally have a decreased content of uncharged polar amino acids (i.e., Q, N, S, and T) and the hyperthermophilic proteins have an increased content of charged amino acid residues (i.e., K, E, and R) (4,5,16,42,48). As S and T can catalyze the deamination and backbone cleavage of Q and N residues (5, 40), a reduction in the amounts of all four of these residues would minimize deamination.…”