An Unusual Mechanism of Glycoside Hydrolysis Involving Redox and Elimination Steps by a Family 4 β-Glycosidase from <i>Thermotoga maritima</i>

Yip, Vivian L. Y.; Varrot, A.; Davies, G.J.; Rajan, Shyamala S.; Yang, Xiaojing; Thompson, John F.; Anderson, W.F.; Withers, Stephen G.

doi:10.1021/ja047632w

Cited by 133 publications

(183 citation statements)

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“…13 Current understanding of glycosidase mechanisms has been reviewed thoroughly, [14][15][16][17] and recently three new general concepts have been put forward: 1) the syn/anti proton donor positioning; 12 2) the presence of a hydrophobic platform as a common feature within the -1 subsite; 18 and 3) enzymes with syn-positioned proton donors perform electrostatic transition state (TS) stabilization (known to be the most important factor for enzymatic catalysis 19,20 ) towards the substrate's ring oxygen by means of the conjugate base of their proton donor, whereas antiprotonating enzymes provide a separate electron-rich residue for this purpose. 21 The considerations in this work pertain to pyranoside glycosidic bond substitutions that operate by a classical exocyclic mechanism on oxyalkyl-type leaving groups, thus not to: 1) the endocyclic mechanism 22 which has not yet been observed with glycoside hydrolases; 2) the SN isubstitution mechanism that occurs in glycosyl fluoride solvolysis 23 and in-situ anomerisations with good leaving groups, 24 and has been proposed to be operative in retaining glycosyltransferases; 25 3) catalysis by GH family 4 (GH4) glycosidases that operate by a redox and elimination mechanism; 26 and 4) GH families 18, 20 (Clan K) 27,28 and 84 29 that utilize anchimeric assistance from the substrates' N-acetyl group, resulting in a strained oxazoline-type glycosyl intermediate (chitinases/chitobiases). Based on its orientation relative to a reference atom in the Fisher projection (IUPAC nomenclature), an α/β glycosidic bond often -but not always -corresponds with an axial/equatorial substituent orientation in the ground state conformation.…”

Section: Introductionmentioning

confidence: 99%

Itineraries of enzymatically and non-enzymatically catalyzed substitutions at O-glycopyranosidic bonds

Nerinckx¹,

Desmet²,

Claeyssens³

2006

Arkivoc

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Several crystal structures of glycoside hydrolases in complex with a substrate analog, or of inactive mutants complexed with a substrate, reveal non-ground state carbohydrate conformations within subsite -1 of the active site. These "frozen" local minima as preferred by the enzyme represent pre-transition-state situations along the reaction itinerary. Substantiated by theoretical considerations, this leads to the proposal that substitutions on β-equatorial as well as α-axial D-O-glycopyranosidic bonds may always follow an itinerary that is predetermined by the original configuration at the anomeric center, where the formation of a transition state that is similar to a half-chair is always preceded by a conformational change away from the ground state.

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Section: Introductionmentioning

confidence: 99%

Itineraries of enzymatically and non-enzymatically catalyzed substitutions at O-glycopyranosidic bonds

Nerinckx¹,

Desmet²,

Claeyssens³

2006

Arkivoc

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“…Equally perplexing was the finding that the dinucleotide cofactor was not consumed during hydrolysis, and no NADH was formed at the end of the catalytic cycle. It was only in 2004, after the solution of the structures of the first GH4 Pagl (GlvA from B. subtilis; Protein Data Bank code 1u8x) and phospho-␤-glucosidase (BglT from Thermotoga maritima; Protein Data Bank code 1up6) and extensive kinetic analyses, that a catalytic mechanism could be proposed for these unique enzymes (28,40,(47)(48)(49). In this mechanism, hydrolysis proceeds via a sequence of oxidation-eliminationaddition and reduction reactions, which result in overall retention of the configuration at the anomeric center.…”

Section: Discussionmentioning

confidence: 99%

ThesimOperon Facilitates the Transport and Metabolism of Sucrose Isomers inLactobacillus caseiATCC 334

Thompson¹,

Jakubovics²,

Abraham

et al. 2008

J Bacteriol

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Inspection of the genome sequence of Lactobacillus casei ATCC 334 revealed two operons that might dissimilate the five isomers of sucrose. To test this hypothesis, cells of L. casei ATCC 334 were grown in a defined medium supplemented with various sugars, including each of the five isomeric disaccharides. Extracts prepared from cells grown on the sucrose isomers contained high levels of two polypeptides with M r s of ϳ50,000 and ϳ17,500. Neither protein was present in cells grown on glucose, maltose or sucrose. Proteomic, enzymatic, and Western blot analyses identified the ϳ50-kDa protein as an NAD ؉ -and metal ion-dependent phospho-␣-glucosidase. The oligomeric enzyme was purified, and a catalytic mechanism is proposed. The smaller polypeptide represented an EIIA component of the phosphoenolpyruvate-dependent sugar phosphotransferase system. Phospho-␣-glucosidase and EIIA are encoded by genes at the LSEI_0369 (simA) and LSEI_0374 (simF) loci, respectively, in a block of seven genes comprising the sucrose isomer metabolism (sim) operon. Northern blot analyses provided evidence that three mRNA transcripts were up-regulated during logarithmic growth of L. casei ATCC 334 on sucrose isomers. Internal simA and simF gene probes hybridized to ϳ1.5-and ϳ1.3-kb transcripts, respectively. A 6.8-kb mRNA transcript was detected by both probes, which was indicative of cotranscription of the entire sim operon.Comparative genomics and phylogenetic analyses of the lactic acid bacteria (LAB) have provided evidence that the order Lactobacillales comprises the families Lactobacillaceae, Streptococcaceae, Enterococcaceae, and Leuconostocaceae (17, 25; http://www.ncbi.nlm.nih.gov/Taxonomy/). These generally fastidious gram-positive organisms are used extensively for the fermentation of dairy, meat, and vegetable products. The industrial importance of LAB, and Lactobacillus casei strains in particular, results from the capacity of these microorganisms to metabolize carbohydrates rapidly (and primarily) to lactic acid. Prior to their homolactic fermentation via the glycolytic pathway, many carbohydrates are accumulated simultaneously with phosphorylation via sugar-specific phosphoenolpyruvate (PEP)-dependent phosphotransferase systems (PTSs) (7,24,29). The multicomponent PEP-dependent PTSs comprise membrane-localized, sugar-specific transporters (IICB enzymes) that may be fused or associated with a third protein (EIIA), as well as two general cytoplasmic proteins (EI and HPr). Collectively, these interactive proteins constitute a fivestage phosphorelay that, via transfer of the high-energy phosphoryl moiety from PEP, catalyzes the simultaneous phosphorylation and translocation of sugars through the cytoplasmic membrane. Biochemical and physiological studies performed during the past 25 years have established the presence of a variety of sugar PEP-dependent PTSs in L. casei, including PTSs for glucose (41, 45), galactose (2, 6), lactose (4,5,8,9), sorbose (46), and pentitols (15, 16). Significantly, in the past decade, the development ...

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“…From these data, it is clear that when transformed with plasmid pAP2 (encoding aglA and aglB), MG-1655 acquires the additional capacity to metabolize not only ␣-MGlc but also maltitol, isomaltose, and all five isomers of sucrose. Importantly, the detection of AglA-specific peptides by LC- a series of oxidation, elimination, addition, and reduction steps (42)(43)(44)(45), in a reaction mechanism unanticipated during the past 50 years of research on glycoside hydrolysis. Amino acids at the active site of GlvA (shown in boldface) are positionally conserved in AglB as follows: Ser-15 (13) AglB with 1U8X as template shows that the structures of the two phospho-␣-glucosidases are virtually identical.…”

Section: Discussionmentioning

confidence: 99%

Genetic Requirements for Growth of Escherichia coli K12 on Methyl-α-D-glucopyranoside and the Five α-D-Glucosyl-D-fructose Isomers of Sucrose

Pikis

Hess

Arnold

et al. 2006

Journal of Biological Chemistry

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Strains of For almost 50 years, methyl-␣-D-glucopyranoside (␣-MGlc)3 has served as a model substrate for probing the mechanism(s) of sugar transport in bacteria. Fortuitously (but importantly), ␣-MGlc is not metabolized by Escherichia coli K12, and transport data were interpretable without the ambiguities caused by further catabolism of the accumulated sugar. Early studies of ␣-MGlc uptake by cells of E. coli introduced the "permease" concept to bacterial physiologists, and "active transport" was incorporated in the lexicon of microbial energetics (1-5). The toxicity of ␣-MGlc toward E. coli was soon recognized, and Rogers and Yu (2) unexpectedly found that the glucose analog was recoverable from cells in both free and phosphorylated form. An understanding of these observations awaited the serendipitous discovery, and subsequent biochemical dissection, of the bacterial phosphoenolpyruvate-dependent sugar:phosphotransferase system (PEP-PTS; EC 2.7.1.69) by Roseman and co-workers (6, 7). The multicomponent PEP-PTS includes membrane-localized, sugar-specific transporters (enzymes IICB) that are fused or associated with a third domain (IIA) and two general cytoplasmic proteins (enzyme I and HPr). Collectively, these interactive components constitute a phospho-relay that (in five sequential stages) transfers the high energy phosphoryl moiety from PEP to catalyze the simultaneous phosphorylation and translocation of sugars through the cytoplasmic membrane (for reviews of PTS functions and nomenclature see . Completion of the chromosomal DNA sequence of E. coli K12 strain MG-1655 in 1997 (15), confirmed that ptsG encodes one (IICB Glc /PtsG) of two glucose-specific PTS transporters, whereas the genes for the general proteins and IIA Glc reside within a separate ptsHIcrr operon. After the discovery of other PEP-PT systems, it became apparent that ␣-MGlc was a reasonably specific substrate for the PtsG transporter in E. coli. , it was established that ␣-MGlc6P was accumulated via PtsG to toxic concentrations and that intracellular ␣-MGlc was formed via dephosphorylation of ␣-MGlc6P (Fig. 1, left).Our interest in the transport and bactericidal effects of ␣-MGlc(6P) in E. coli K12 stems from the fact that Klebsiella pneumoniae ATCC 23357 (a taxonomically close relative) readily ferments ␣-MGlc as an energy source for growth. Furthermore, and again in contrast with E. coli K12 strains, K. pneumoniae also metabolizes the following five O-␣-linked isomers of sucrose: trehalulose, turanose, maltulose, leucrose, and palatinose (20, 21). We presented evidence (21) for a tricistronic ␣-glucoside (Agl) operon comprising genes aglR, aglA, and aglB. We suggested that the three genes encode (in order), a transcriptional regulatory protein (AglR), an ␣-glucoside PTS transporter AglA (IICB Agl Swiss-Prot accession number Q9AGA7), and an Mn

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An Unusual Mechanism of Glycoside Hydrolysis Involving Redox and Elimination Steps by a Family 4 β-Glycosidase from Thermotoga maritima

Cited by 133 publications

References 23 publications

Itineraries of enzymatically and non-enzymatically catalyzed substitutions at O-glycopyranosidic bonds

Itineraries of enzymatically and non-enzymatically catalyzed substitutions at O-glycopyranosidic bonds

ThesimOperon Facilitates the Transport and Metabolism of Sucrose Isomers inLactobacillus caseiATCC 334

Genetic Requirements for Growth of Escherichia coli K12 on Methyl-α-D-glucopyranoside and the Five α-D-Glucosyl-D-fructose Isomers of Sucrose

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