Crystal structure of the reduced Schiff‐base intermediate complex of transaldolase B from <i>escherichia coli</i>: Mechanistic implications for class I aldolases

Jia, Jia; Schörken, Ulrich; Lindqvist, Ylva; Sprenger, Georg A.; Schneider, Günter

doi:10.1002/pro.5560060113

Cited by 72 publications

(80 citation statements)

References 21 publications

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“…This topology was consistent with the typical core structure among the superfamily of transaldolases. 4,6,20 A homodimer of NQM1/YGR043C [ Fig. 1(B)] was observed in crystal, as well as in solution demonstrated by size exclusion chromatography.…”

Section: Resultsmentioning

confidence: 99%

“…NQM1/ YGR043C contained all residues that are highly conserved and invariant in the transaldolase superfamily, 4 for instance, the Lys on b4 strand of the core a/b barrel, where the reduced Schiff-base intermediate is produced. 6 Three dimensional structure comparisons revealed that both the overall structure and the core a/b barrel of NQM1/YGR043C are similar to those in other transaldo- Numbers in parentheses are for the highest resolution shell. a R merge 5 P jI i 2 I m j/ P I i , where I i is the entensity of the measured reflection and I m is the mean intensity of all symmetry-related reflections.…”

Section: Comparison With Other Transaldolasesmentioning

confidence: 92%

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The crystal structure and identification of NQM1/YGR043C, a transaldolase from Saccharomyces cerevisiae

Huang

Hui

et al. 2008

Proteins

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Section: Resultsmentioning

confidence: 99%

Section: Comparison With Other Transaldolasesmentioning

confidence: 92%

The crystal structure and identification of NQM1/YGR043C, a transaldolase from Saccharomyces cerevisiae

Huang

Hui

et al. 2008

Proteins

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“…Transaldolases that have the same fold and similar active site disposition of catalytic residues as class I aldolases (16) differ by the exceptional stability of their enamine (45) due to an absence of a mechanistic pathway enabling interconversion of the enamine to the iminium. Indeed, in contrast to class I aldolase, the active site of bacterial transaldolase B has no amino acid residue capable of charge stabilization vicinal to the C3 carbon of dihydroxyacetone and the enzyme lacks a mobile C-terminal region capable of mediating ␣-proton transfer (46).…”

Section: Discussionmentioning

confidence: 99%

Stereospecific Proton Transfer by a Mobile Catalyst in Mammalian Fructose-1,6-bisphosphate Aldolase

St-Jean¹,

Sygusch

2007

Journal of Biological Chemistry

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Class I fructose-1,6-bisphosphate aldolases catalyze the interconversion between the enamine and iminium covalent enzymatic intermediates by stereospecific exchange of the pro(S) proton of the dihydroxyacetone-phosphate C3 carbon, an obligatory reaction step during substrate cleavage. To investigate the mechanism of stereospecific proton exchange, high resolution crystal structures of native and a mutant Lys 146 Stereospecificity is one of the hallmarks of enzyme catalysis. Aldolases, which are abundant and ubiquitous enzymes, catalyze stereospecific carbon-carbon bond formation, one of the most important transformations in living organisms. Their role is best known in glycolysis where fructose-1,6-bis(phosphate) (FBP) 3 aldolases (EC 4.1.2.13) promote the cleavage of FBP to triose phosphates, D-glyceraldehyde-3-phosphate (G3P), and dihydroxyacetone phosphate (DHAP). A common feature to class I enzymes is the use of a covalent mechanism for catalysis implicating iminium (protonated Schiff base) formation between a lysine residue on the enzyme and a ketose substrate (1) that entails stereospecific proton exchange in the covalent intermediate (2). Of the three aldolase isozymes found in vertebrates (3), the catalytic mechanism has been extensively studied using class I aldolase A from rabbit muscle and key intermediates are depicted in reaction Scheme 1.In the condensation direction, the reaction involves covalent intermediate formation with the keto triose phosphate DHAP followed by condensation with the aldehyde G3P to form the ketose of the acyclic FBP substrate (4, 5). To form the C3-C4 bond of FBP, the enzyme stereospecifically abstracts the pro(S) C3 proton of the trigonal iminium 1 (6, 7) that is formed from the Michaelis complex with DHAP thereby generating via the enamine 2 (2) the carbanionic character at C3 of DHAP for the aldol reaction. The nascent carbon-carbon bond has the same orientation as the pro(S) ␣-hydrogen initially abstracted from the DHAP imine intermediate. The overall retention of configuration at C3 requires that proton abstraction from 1 to yield the enamine 2 and condensation with aldehyde in 3 must take place from the same direction on the enzyme (8). The iminium intermediate formed is then hydrolyzed and FBP is released by the inverse reaction sequence shown in Scheme 1.A distinguishing mechanistic feature of class I aldolases is the relative stability of the iminium 1 and enamine 2 forms, which is a consequence of the catalytic requirements. The enzyme must stabilize the enamine 2 and/or the preceding iminium 1 such that no decomposition occurs prior to reaction with the aldehyde as shown in 3. This stability is reflected in solution where the enzymatic populations 1 and 2 represent 20 and 60%, respectively, of bound DHAP on the muscle enzyme under equilibrium conditions (9). The interconversion between the two forms implicates the conserved C-terminal Tyr 363 residue whose proteolysis inhibits the stereospecific proton exchange step, making it rate-limiting (10), whereas the pen...

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“…The best studied example is transaldolase B (TalB) from Escherichia coli (1). A number of structural and mechanistic studies have been published elucidating its reaction mechanism (1)(2)(3)(4)(5)(6). Similar to class I aldolases the reaction proceeds via a Schiff base intermediate.…”

Section: Transaldolase (Tal)mentioning

confidence: 99%

“…In TalB, Phe 178 cannot form an H-bond with the water molecule. Hence, the catalytically important water molecule is only coordinated by 2 residues, Glu 96 and Thr 156 (6) (Fig. 6).…”

mentioning

confidence: 99%

Replacement of a Phenylalanine by a Tyrosine in the Active Site Confers Fructose-6-phosphate Aldolase Activity to the Transaldolase of Escherichia coli and Human Origin

Schneider

Sandalova

Schneider

et al. 2008

Journal of Biological Chemistry

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Based on a structure-assisted sequence alignment we designed 11 focused libraries at residues in the active site of transaldolase B from Escherichia coli and screened them for their ability to synthesize fructose 6-phosphate from dihydroxyacetone and glyceraldehyde 3-phosphate using a newly developed color assay. We found one positive variant exhibiting a replacement of Phe 178 to Tyr. This mutant variant is able not only to transfer a dihydroxyacetone moiety from a ketose donor, fructose 6-phosphate, onto an aldehyde acceptor, erythrose 4-phosphate (14 units/mg), but to use it as a substrate directly in an aldolase reaction (7 units/mg). With a single amino acid replacement the fructose-6-phosphate aldolase activity was increased considerably (>70-fold compared with wild-type). Structural studies of the wild-type and mutant protein suggest that this is due to a different H-bond pattern in the active site leading to a destabilization of the Schiff base intermediate. Furthermore, we show that a homologous replacement has a similar effect in the human transaldolase Taldo1 (aldolase activity, 14 units/mg). We also demonstrate that both enzymes TalB and Taldo1 are recognized by the same polyclonal antibody. Transaldolase (Tal)2 is a ubiquitous enzyme that is present in all domains of life. It is part of the non-oxidative path of the pentose phosphate pathway. Here, it catalyzes the reversible transfer of a dihydroxyacetone (DHA) moiety from a ketose donor, e.g. fructose 6-phosphate (Fru-6-P), onto an aldehyde acceptor, e.g. erythrose 4-phosphate (Ery-4-P). The best studied example is transaldolase B (TalB) from Escherichia coli (1). A number of structural and mechanistic studies have been published elucidating its reaction mechanism (1-6). Similar to class I aldolases the reaction proceeds via a Schiff base intermediate. TalB is a homo-dimer and the monomer exhibits a (␤/␣) 8 -barrel fold where the C-terminal helix lies across the barrel opening at one site (5). A similar structure had been determined for the human transaldolase (7).Recently, a fructose-6-phosphate aldolase (FSA) of E. coli has been discovered by our group (8) that uses DHA as donor substrate and catalyzes the reversible formation of Fru-6-P from DHA and GAP (Fig. 1). Multiple sequence alignments of different Tal sequences demonstrate that FSA resides within the family of transaldolases (8, 9) and does show little similarity to DHAP-dependent aldolases, such as class I fructose-1,6-bisphosphate aldolase. Also the FSA monomer is highly similar to TalB exhibiting a similar (␤/␣) 8 -barrel fold (9). But in contrast to TalB, FSA forms a homo-decamer, which is arranged by two doughnut-shaped pentameric rings where the C-terminal helix of one subunit interacts with the adjacent subunit (9). With 220 amino acids compared with 317 amino acids FSA is smaller than TalB and shows about 20% sequence identity to TalB. Mechanistically, it is a class I aldolase, i.e. the reaction proceeds via a Schiff base intermediate.In this study, we addressed the differences b...

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Crystal structure of the reduced Schiff‐base intermediate complex of transaldolase B from escherichia coli: Mechanistic implications for class I aldolases

Cited by 72 publications

References 21 publications

The crystal structure and identification of NQM1/YGR043C, a transaldolase from Saccharomyces cerevisiae

The crystal structure and identification of NQM1/YGR043C, a transaldolase from Saccharomyces cerevisiae

Stereospecific Proton Transfer by a Mobile Catalyst in Mammalian Fructose-1,6-bisphosphate Aldolase

Replacement of a Phenylalanine by a Tyrosine in the Active Site Confers Fructose-6-phosphate Aldolase Activity to the Transaldolase of Escherichia coli and Human Origin

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