The Family 1 β-Glucosidases from Pyrococcus furiosus and Agrobacterium faecalis Share a Common Catalytic Mechanism

Abstract

Comparisons of catalytic mechanisms have not previously been performed for homologous enzymes from hyperthermophilic and mesophilic sources. Here, the beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus was recombinantly produced in Escherichia coli and shown to have biophyscial and biochemical properties identical to those of the wild-type enzyme. Moreover, the recombinant enzyme was subjected to a detailed kinetic investigation at 95 degreesC to compare its catalytic mechanism to that determined at 37 degreesC for the beta-glucosidase (abg) from the mesophilic bacterium, Agrobacterium faecalis [Kempton, J., and Withers, S. G. (1992) Biochemistry 31, 9961]. These enzymes have amino acid sequences that are 33% identical and have been classified as family 1 glycosyl hydrolases on the basis of amino acid sequence similarities. Both enzymes have similar broad specificities for both sugar and aglycone moieties and exhibit nearly identical pH dependences for their kinetic parameters with several different substrates. Bronsted plots were constructed for bgl at several temperatures using a series of aryl glucoside substrates. These plots were concave downward at all temperatures, indicating that bgl utilized a two-step mechanism similar to that of abg and that the rate-limiting step in this mechanism did not change with temperature for any given aryl glucoside. The Bronsted coefficient for bgl at 95 degreesC (beta1g = -0.7) was identical to that for abg at 37 degreesC and implies that these enzymes utilize nearly identical transition states, at least in regard to charge accumulation on the departing glycosidic oxygen. In addition, a high correlation coefficient (rho = 0.97) for the linear free energy relationship between these two enzymes and similar inhibition constants for these two enzymes with several ground state and transition state analogue inhibitors further indicate that these enzymes stabilize similar transition states. The mechanistic similarities between these two enzymes are noteworthy in light of the large difference in their temperature optima. This suggests that, in the presumed evolution that occurred between the hyperthermophilic archaeal enzyme and the mesophilic bacterial enzyme, structural modifications must have been selected which maintained the integrity of the active site structure and, therefore, the specificity of transition state interactions, while adapting the overall protein structure to permit function at the appropriate temperature.

Growth of Hyperthermophilic Archaeon Pyrococcus furiosus on Chitin Involves Two Family 18 Chitinases

“…Kinetic parameters for ChiB cat on pNp-chitooligosaccharides and chitooligosaccharides are summarized in Table 1. For the pNp-linked substrates studied, the highest k cat /K m was observed for pNpchitotetraose, and the highest catalytic efficiency (k cat /K m ) was noted on (GlcNAc) 4 and (GlcNAc) 5 for the series of chitooligosaccharides. Initial rates of chitooligosaccharide hydrolysis showed that the enzyme exhibited Michaelis-Menten kinetics with a DP of Ն3, although significant substrate inhibition was noted on oligosaccharides with a DP of 4 to 6 at concentrations of Ն0.8 mM (Fig.…”

“…Although this chitinase is not closely related to ChiB cat , the specific activity and turnover numbers (k cat ) of ChiB cat are 2 orders of magnitude higher than for the mesophilic organism. Both enzymes showed similar substrate specificity (k cat /K m ) on (GlcNAc) 5 and (GlcNAc) 6 . However, ChiB cat exhibited an order of magnitude higher specific activity on (GlcNAc) 4 than the V. furnissii endochitinase ( Table 1), indicating that a tetrasaccharide optimally binds to the active site of ChiB cat .…”

“…P. furiosus has been found to grow on a variety of ␣-linked carbohydrates, such as starch and maltose (7), as well as on ␤-linked glucan substrates, such as cellobiose (37), laminarin (29), barley ␤-glucan, and some soluble forms of cellulose (5,16). Glycosyl hydrolases from P. furiosus that have been identified and characterized include an ␣-glucosidase (12), two ␣-amylases (35,41,42), an amylopullulanase (8,15), a ␤-glucosidase (37), a ␤-mannosidase (3), a laminarinase (29), and an endoglucanase (4).…”

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

“…Otherwise, hyperthermophilic and mesophilic enzymes are highly similar: (i) the sequences of homologous hyperthermophilic and mesophilic proteins are typically 40 to 85% similar (79,350); (ii) their three-dimensional structures are superposable (16,63,143,160,227,284,327); and (iii) they have the same catalytic mechanisms (22,350,386).…”

Regulation of Endo-Acting Glycosyl Hydrolases in the Hyperthermophilic Bacterium Thermotoga maritima Grown on Glucan- and Mannan-Based Polysaccharides

“…The synergistic activities of several of these enzymes may be needed to degrade polysaccharides in vitro. In combination, ␤-glucosidase (Cel1) (4,30), laminarinase (Lam16) (24), and endoglucanase (Cel13) (2) from P. furiosus rapidly hydrolyze barley ␤-glucan to smaller oligosaccharides and ultimately glucose (15). Because two endo-acting glycosidases appear to be secreted by P. furiosus, as shown by the presence of putative signal peptides, and the ␤-glucosidase is cytoplasmic, an oligosaccharide transport system must also be involved in the utilization of barley ␤-glucan as a carbon and energy source.…”