Extremely Thermostable Serine-type Protease fromAquifex pyrophilus

Abstract

A gene encoding a serine-type protease has been cloned from Aquifex pyrophilus using a sequence tag containing the consensus sequence of proteases as a probe. Sequence analysis of the cloned gene reveals an open reading frame of 619 residues that has three canonical residues (Asp-140, His-184, and Ser-502) that form the catalytic site of serine-type proteases. The size of the mature form (43 kDa) and its localization in the cell wall fraction indicate that both the NH 2 -and COOHterminal sequences of the protein are processed during maturation. When the cloned gene is expressed in Escherichia coli, it is weakly expressed as active and processed forms. The pH optimum of this protease is very broad, and its activity is completely inactivated by phenylmethylsulfonyl fluoride. The half-life of the protein is 6 h at 105°C, suggesting that it is one of the most heat-stable proteases. The cysteine residues in the mature form may form disulfide bonds that are responsible for the strong stability of this protease, because the thermostability of the protein is significantly reduced in the presence of reducing reagent.

Partial Characterization of an Enzyme Fraction with Protease Activity Which Converts the Spore Peptidoglycan Hydrolase (SleC) Precursor to an Active Enzyme during Germination of Clostridium perfringens S40 Spores and Analysis of a Gene Cluster Involved in the Activity

“…The percentage of charged amino acids (Asp, Glu, Arg, and Lys) in each GSP protein is over 20%. It has been shown that salt bridges between charged amino acids are a major factor stabilizing protein structure (7). It is likely, therefore, that intermolecular electrostatic interactions contribute to the formation of the active multiprotein enzyme with a relatively high thermal stability.…”

Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

“…This observation indirectly suggests that the disulfide bridges present in the native enzyme are stabilizing (52). An Aquifex pyrophilus serine protease was recently described that contains eight cysteines (none are present in subtilisin BPN') (64). A dithiothreitol treatment reduced its t 1/2 at 85°C from 90 h to less than 2 h. This destabilization by dithiothreitol at high temperature suggests that this enzyme indeed contains disulfide bridges and that they are highly inaccessible.…”

Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus

“…The presence of disulfide bonds within several archaeal and thermophilic genomes has been postulated, taking into account the results of a recent computational study based on the combination of genomic data with protein structure [13]. Moreover, the increasing number of solved crystallographic structures has highlighted the presence of disulfide bonds in several hyperthermophilic proteins [10][11][12]14]. The results reported here on multiple disulfide bonds in PfMTAP add a new example, to the few present in the literature, of an intracellular hyperthermophilic protein with disulfide bonds and argue strongly that intracellular disulfides could represent a significant mechanism to achieve superior levels of thermostability.…”

“…In recent years, growing attention has been paid to the presence of disulfide bonds in intracellular hyperthermophilic proteins where these covalent links may play a key role in protein stabilization in the extreme thermal environment [10][11][12][13][14].…”

“…Several mechanisms of thermal stabilization have been proposed, among which additional networks of salt bridges and hydrogen bonds, improved packing density and enhanced secondary structure are the most cited [2][3][4][7][8][9]. In spite of this, no general rules have been established to date, and it has been concluded that each protein evolves individually through a limited number of factors that occur at different levels, also involving the amino acid sequence and the quaternary structure of the proteins.In recent years, growing attention has been paid to the presence of disulfide bonds in intracellular hyperthermophilic proteins where these covalent links may play a key role in protein stabilization in the extreme thermal environment [10][11][12][13][14].5¢-Methylthioadenosine phosphorylase (MTAP) catalyzes the reversible phosphorolysis of 5¢-methylthioadenosine (MTA), a sulfur-containing nucleoside formed from S-adenosylmethionine (AdoMet) via several independent pathways of which the polyamine biosynthesis is quantitatively the most important [15]. The products of the MTA cleavage reaction are adenine and 5-methylthioribose-1-phosphate.…”