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

Abstract

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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“…Although the original screening of the TalB mutant libraries had been performed for their ability to synthesize fructose 6-phosphate (Fru6P) from DHA and GA3P, [20] the latter was dismissed as a potential reference component for judging synthetic applications because of its inherent instability towards decomposition. [26] By using DHA as a donor, TalB F178Y indeed produced a single product, as indicated by a new spot on TLC that was subsequently identified as d-fructose 1 a by its typical sets of NMR signals (Scheme 2).…”

“…Comparison of the X-ray structures of the active site of FSA (dark gray; pdb 1L6w) [23] and TalB F178Y (light gray; pdb 3cwn). [20] The figure was prepared by using PyMOL. [25] www.chemeurj.org boligations.…”

“…As a first result of our joint program focusing on the directed evolution of transaldolase B from E. coli (TalB) [19] towards novel specificities, very recently some of us reported that replacement of a single amino acid residue, phenylalanine 178, by tyrosine in the active site confers true aldolase, instead of transaldolase, activity to the mutant enzyme (TalB F178Y ; Scheme 1). [20] Herein we report on the donor and acceptor substrate space of this engineered TalB F178Y mutein in comparison with the related wild-type fructose 6-phosphate aldolase (FSA) from E. coli, another member of the transaldolase class.…”

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“…An overlay of the two structures (SI Methods) reveals a superimposable α 8 /β 8 -barrel including the conserved active site residues (Fig. S3C) previously demonstrated to be important in catalysis (31,32). The major distinguishing feature between these transaldolases is the number and orientation of exterior α-helices, with TalB containing more external α-helices relative to TalC's more compact structure.…”

“…S3A) but no change in the active site residues essential for catalysis ( Fig. S3A) (31,32). TalC more closely resembles the Escherichia coli fructose-6-phosphate (F6P) aldolase, FsaA (33), than host TalB (Fig.…”