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␣-L-Arabinofuranosidases cleave the L-arabinofuranoside side chains of different hemicelluloses and are key enzymes in the complete degradation of the plant cell wall. The ␣-L-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase, was subjected to a detailed mechanistic study. Aryl-␣-Larabinofuranosides with various leaving groups were synthesized and used to verify the catalytic mechanism and catalytic residues of the enzyme. The steady-state constants and the resulting Brønsted plots for the E175A mutant are consistent with the role of Glu-175 as the acid-base catalytic residue. The proposed nucleophile residue, Glu-294, was replaced to Ala by a double-base pairs substitution. The resulting E294A mutant, with 4-nitrophenyl ␣-L-arabinofuranoside as the substrate, exhibited eight orders of magnitude lower activity and a 10-fold higher K m value compared with the wild type enzyme. Sodium azide accelerated by more than 40-fold the rate of the hydrolysis of 2 ,4 ,6 -trichlorophenyl ␣-Larabinofuranoside by the E294A mutant. The glycosylazide product formed during this reaction was isolated and characterized as ␤-L-arabinofuranosyl-azide by 1 H NMR, 13 C NMR, mass spectrometry, and Fourier transform infrared analysis. The anomeric configuration of this product supports the assignment of Glu-294 as the catalytic nucleophile residue of the ␣-L-arabinofuranosidase T-6 and allows for the first time the unequivocal identification of this residue in glycoside hydrolases family 51.␣-L-Arabinofuranosidases (EC 3.2.1.55) are hemicellulases that cleave the glycosidic bond between L-arabinofuranosides side chains and different oligosaccharides. These enzymes are part of an array of glycoside hydrolases responsible for the degradation of hemicelluloses such as arabinoxylan, arabinogalactan, and L-arabinan (1-3). The L-arabinofuranoside substitutions on xylans can strongly inhibit the action of endoxylanases and ␤-xylosidases, thus preventing the complete degradation of the polymer to its basic xylose units (4, 5). In many cases, microorganisms that utilize hemicelluloses possess ␣-L-arabinofuranosidases with various substrate specificities and biochemical properties (6). To date, there are more than 110 sequences of different ␣-L-arabinofuranosidases, which are classified, based on sequence homology, into four glycoside hydrolase families (GHs) 1 : GH43, GH51, GH54, and GH62 (7,8). Hemicellulases, together with cellulases, have a key role in the carbon cycle in nature, because they are responsible for the complete degradation of the plant biomass to soluble saccharides. These in turn can be used as carbon or energy sources for microorganisms and higher animals. Hemicellulases have attracted much attention in recent years because of their potential industrial use in biobleaching of paper pulp, bioconversion of lignocellulose material to fermentative products, improvement of animal feedstock digestibility, and organic synthesis (6, 9 -11).The glycosidic bond is one of the most stable bonds in...

Section: Resultsmentioning

confidence: 99%

Detailed Kinetic Analysis and Identification of the Nucleophile in α-l-Arabinofuranosidase from Geobacillus stearothermophilus T-6, a Family 51 Glycoside Hydrolase

Shallom¹,

Belakhov²,

Solomon³

et al. 2002

“…Such a focus of productive mutations upon improving transition state interactions with the C2-hydroxyl group is perhaps unsurprising because our earlier studies regarding this and other ␤-glucosidases have shown that interactions at the 2-position can contribute up to 10 kcal/mol to transition state binding (41,(43)(44)(45). Furthermore, structural studies on several of these enzymes had revealed that the most important contribution to such interactions was in fact the carbonyl oxygen of the nucleophile (45)(46)(47). Creation of the glycosynthase by mutation of Glu-358 thereby not only removes the ability to form a glycosyl-enzyme intermediate, but also removes a key stabilizing interaction to the substrate C2-hydroxyl group.…”

Section: Discussionmentioning

confidence: 99%

Directed Evolution of a Glycosynthase from Agrobacterium sp. Increases Its Catalytic Activity Dramatically and Expands Its Substrate Repertoire

Kim

Lee

Warren

et al. 2004

122

The Agrobacterium sp. ␤-glucosidase (Abg) is a retaining ␤-glycosidase and its nucleophile mutants, termed Abg glycosynthases, catalyze the formation of glycosidic bonds using ␣-glycosyl fluorides as donor sugars and various aryl glycosides as acceptor sugars. Two rounds of random mutagenesis were performed on the best glycosynthase to date (AbgE358G), and transformants were screened using an on-plate endocellulase coupled assay. Two highly active mutants were obtained, 1D12 (A19T, E358G) and 2F6 (A19T, E358G, Q248R, M407V) in the first and second rounds, respectively. Relative catalytic efficiencies (k cat /K m ) of 1:7:27 were determined for AbgE358G, 1D12, and 2F6, respectively, using ␣-D-galactopyranosyl fluoride and 4-nitrophenyl ␤-D-glucopyranoside as substrates. The 2F6 mutant is not only more efficient but also has an expanded repertoire of acceptable substrates. Analysis of a homology model structure of 2F6 indicated that the A19T and M407V mutations do not interact directly with substrates but exert their effects by changing the conformation of the active site. Much of the improvement associated with the A19T mutation seems to be caused by favorable interactions with the equatorial C2-hydroxyl group of the substrate. The alteration of torsional angles of Glu-411, Trp-412, and Trp-404, which are components of the aglycone (؉1) subsite, is an expected consequence of the A19T and M407V mutations based on the homology model structure of 2F6.Oligosaccharides have considerable potential as therapeutics because of the numerous medicinally relevant physiological events that involve glycoconjugates (1-3). To expand our understanding of the various roles of oligosaccharides found in important cellular events, more efficient and selective synthetic protocols must be developed for the preparation of oligosaccharides. Classical chemical synthesis is often impractical for the synthesis of complex oligosaccharides because of the need for selective and labor-intensive protection-deprotection steps and difficulties in directing product stereochemistry. To overcome these limitations, enzymatic syntheses using glycosidases or glycosyl transferases have rapidly gained prominence (4 -6).In recent years, the glycosynthase approach developed in this laboratory has added a new dimension to the enzymatic preparation of oligosaccharides (7-9). Glycosynthases are retaining glycosidase mutants in which the catalytic nucleophile has been converted to a non-nucleophilic residue. These mutants catalyze the formation of glycosidic bonds when glycosyl fluorides with anomeric configuration opposite to that of the original substrate, thereby mimicking the glycosyl enzyme intermediate, are employed as substrates. The modified enzyme catalyzes the nucleophilic displacement of the fluoride via attack by a hydroxyl group on an added glycosyl acceptor, generating a new glycosidic bond with the same stereochemistry as the normal substrate. The reactions catalyzed by glycosynthases are highly amenable to industrial syntheses because of the hig...

“…The existing structural and kinetic evidence for both classes of inverting enzymes supports a direct displacement mechanism involving an S N 2-like attack facilitated by an active site general base catalyst. Reactions catalyzed by retaining glycosidases are known to proceed via a double displacement mechanism involving the formation and subsequent breakdown of a covalent glycosyl-enzyme intermediate (16). Recent findings have, however, brought into question the applicability of this mechanism to retaining nucleoside diphosphate-utilizing glycosyltransferases since good candidates for the catalytic nucleophile are generally lacking, and in no case has an intermediate been trapped and characterized.…”

mentioning

confidence: 99%

Intermediate Trapping on a Mutant Retaining α-Galactosyltransferase Identifies an Unexpected Aspartate Residue

Lairson

Chiu

et al. 2004

Lipopolysaccharyl-␣-1,4-galactosyltransferase C (LgtC), a glycosyltransferase family 8 ␣-1,4-galactosyltransferase from Neisseria meningitidis, catalyzes the transfer of galactose from UDP galactose to terminal lactose-containing acceptor sugars with net retention of anomeric configuration. To investigate the potential role of discrete nucleophilic catalysis suggested by the double displacement mechanism generally proposed for retaining glycosyltransferases, the side chain amide of Gln-189, which is suitably positioned to act as the catalytic nucleophile of LgtC, was substituted with the more nucleophilic carboxylate-containing side chain of glutamate in the hope of accumulating a glycosyl-enzyme intermediate. The resulting mutant was subjected to kinetic, mass spectrometric, and x-ray crystallographic analysis. Although the K m for UDP-galactose is not significantly altered, the k cat was reduced to 3% that of the wild type enzyme. Electrospray mass spectrometric analysis revealed that a steady state population of the Q189E variant contains a covalently bound galactosyl moiety. Liquid chromatographic/mass spectrometric analysis of fragmented proteolytic digests identified the site of labeling not as Glu-189 but, surprisingly, as the sequentially adjacent Asp-190. However, the side chain carboxylate of Asp-190 is located 8.9 Å away from the donor substrate in the available crystal structure. Kinetic analysis of a D190N mutant at this position revealed a k cat value 3000-fold lower than that of the wild type enzyme. A 2.6-Å crystal structure of the Q189E mutant with bound uridine 5 -diphospho-2-deoxy-2-fluoro-␣-D-galactopyranose revealed no significant perturbation of the mode of donor sugar binding nor of active site configuration. This is the first trapping of an intermediate in the active site of a retaining glycosyltransferase and, although not conclusive, implicates Asp-190 as an alternative candidate catalytic nucleophile, thereby rekindling a longstanding mechanistic debate.Oligosaccharides on glycoproteins and glycolipids distributed on cell surfaces and within extracellular matrices are known to play key roles in normal cell functions including cell growth and differentiation, recognition by the immune system, and cell-cell interactions (1-3). Changes in the composition of these glycoconjugates are often associated with disease states, including the metastasis of cancerous cells and autoimmune responses (4 -7). They are also known to modulate interactions with viral and bacterial pathogens leading to infection and are involved in mechanisms to evade host immune responses (8 -10). Glycosyltransferases, the anabolic enzymes responsible for the highly specific construction of these carbohydrate structures, therefore, not only represent an attractive class of therapeutic targets but also are important tools for the enzymatic synthesis of this synthetically challenging class of therapeutic agents. Of central importance to both of these applications is a detailed understanding of the mechanisms by which this c...