Active Site and the Reaction Mechanism of TacTAL

As a member of the class I aldolase family, transaldolase catalyzes its reaction using an active site lysine, which forms the Schiffbase intermediate with the substrate (Venkataraman andRacker, 1961). InTacTAL, this lysine (Lys⁸⁶) is located in theβ₄-strand of the barrel structure. Interestingly, the corresponding lysine residue of the mechanistically related class I fructose-1,6-bis(phosphate) aldolase (FBPA) is located in theβ₆-strand of the barrel (Jiaet al., 1996). In this context, a common ancestor was suggested, which evolved into the present-day aldolases and transaldolases. During the evolution, a circular permutation occurred on gene level resulting in circular permuted TIM-barrel structures. The circular permutation events are suggested to be a common principle in the evolution of proteins belonging to the TIM-barrel family (Naganoet al., 2002) and were assumed to play a role in the evolution of non-TIM-barrel enzymes too, as it was suggested for the 1,3-1,4-β-glucanase (Heinemann andHahn, 1995).

For the reaction ofTacTAL, the reactive lysine in the active site of transaldolase has to be deproto-nated for the nucleophilic attack on the sugar’s carbonyl group (Stellmacheret al., 2015). This is performed by a conserved glutamate residue (Glu⁶⁰inTacTAL). This glutamate is the general

Scheme 2.3: Proposed reaction mechanism of transaldolase/aldolase. The transaldolase reac-tion (TAL) results in the reversible transfer of a dihydroxyacetone unit from ketose phosphates (donor substrate) to the C1 position of the aldose phosphates (acceptor substrate). The aldolase reaction (FSA) results in the reversible cleavage of the ketose-phosphate substrate. Both activities share the first half-reaction (steps 1 – 5) of the catalytic cycle. The main difference between the mechanisms is the protonation of the carbanion/enamine intermediate (Schiffbase in FSA).

Published in Sautneret al. (2015).

acid-base catalyst of transaldolase (Lehwess-Litzmannet al., 2011b). Once deprotonated, the lysine residue can perform the nucleophilic attack on the carbonyl carbon of the donor-substrate, resulting in the formation of the carbinolamine intermediate (scheme 2.3, steps 1 and 2). The carbinolamine’s C2 hydroxy group formed upon the nucleophilic attack and tautomerization step is protonated by the general acid-base catalyst Glu⁶⁰and followed by dehydration (steps 3 and 4).

After water elimination, the Schiffbase intermediate is formed. Deprotonation of the C4 hydroxy group by the glutamate residue and the subsequent rearrangement result in the aldol cleavage of the intermediate (step 5). After product release, the reactive lysine remains modified as covalently linked carbanion/enamine intermediate. This reaction sequence describes the donor half-reaction

of the catalytic cycle. The acceptor half-reaction starts with the binding of the acceptor substrate followed by the nucleophilic attack of the carbanion/enamine intermediate on the carbonyl group of the acceptor substrate. Because of the reversible nature of the catalytic steps, the acceptor half-reaction corresponds to the reverse donor half-reaction.

The proton transfer reactions between the catalytic glutamate and the intermediates are facilitated by the catalytic water molecule observed in the active site, which is conserved in transaldolases (Jia et al., 1997; Thorellet al., 2002). However, the presence of this catalytic water molecule during all catalytic steps is in question (Lehwess-Litzmann, 2011; Tittmann, 2014). This catalytic water molecule is coordinated by the residues Glu⁶⁰and Thr¹¹⁰(figure 2.1.5). In the structure containing the F6P-Schiffbase intermediate in the active site this water is additionally coordinated by the C4 hydroxy group of the intermediate (Lehwess-Litzmannet al., 2011b).

F6P-Schiff base

Asp6 Glu60

Lys86 Thr110

Asn28

W1

Phe132

C4OH Figure 2.1.5: Catalytic water in the active site of TacTAL. The selected active site residues (gray) and the F6P-Schiffbase inter-mediate (yellow) are represented as sticks. The polypeptide backbone (cyan) is shown as sec-ondary structure. The catalytic water molecule (W1, framed) is shown as red sphere. The in-teraction partners of the water are labeled in red.

While most of the active site residues ofTacTAL that are important for the catalysis and/or substrate binding are conserved in the relatedEcFSA (Asp⁶, Asn²⁸, Lys⁸⁶, Thr¹¹⁰, Arg¹³⁵and Ser¹⁶⁷), some important active site residues are different and replaced inEcFSA by hydrophobic (Ser⁵⁸→Phe, Asn¹⁰⁸ → Leu and Ser¹³⁰ → Ala) or homologous residues (Glu⁶⁰ → Gln, Phe¹³² → Tyr and Arg¹⁶⁹→ Lys). The absence of the transaldolase’s general acid-base catalyst Glu⁶⁰ (Gln⁵⁹ in EcFSA) and the presence of a tyrosine residue at the position of Phe¹³²(Tyr¹³¹inEcFSA) are of particular interest. It could be shown that a single mutation of the corresponding phenylalanine residue to tyrosine in the E. colitransaldolase andTacTAL results in a variant with stimulated aldolase activity (Lehwess-Litzmann, 2011; Schneideret al., 2008).

According to the proposed reaction mechanisms (scheme 2.3) ofTacTAL andEcFSA, both enzymes share the first half-reaction (donor half-reaction) of the catalytic cycle. In the case ofTacTAL, those reactions are acid-base catalyzed by the residue Glu⁶⁰. The absence of the equivalent residue in

the active site ofEcFSA is therefore surprising, since the acid-base catalyzed aldol-cleavage of the substrate is also a part of the reaction mechanism of this enzyme. With exception of Asp⁶, only the residue Tyr¹³¹ can perform the acid-base catalysis in the active site ofEcFSA. The Asp⁶is suggested to play an important role in the substrate binding and its correct orientation but does not sufficiently participate on the acid-base catalysis (Lehwess-Litzmannet al., 2011b). In the TacTAL structure containing the F6P-Schiffbase intermediate, this residue coordinates the C3 and C5 hydroxy groups of the intermediate. On this basis, one can assume that the Tyr¹³¹residue takes over the role of the transaldolase’s glutamate residue as the general acid-base catalyst.

The main difference in the catalytic mechanism of FSA compared to the reaction catalyzed by TAL is the protonation of the carbanion/enamine intermediate. In the case of TAL, this intermediate im-mediately reacts with the acceptor substrate (transaldolase reaction), while in FSA, the intermediate is protonated, forming a DHA-Schiffbase intermediate (scheme 2.3). The DHA-Schiffbase cannot attack the acceptor substrate nucleophilically but can be hydrolyzed (aldolase reaction). In this context, the Tyr¹³¹residue inEcFSA is assumed to protonate the carbanion/enamine intermediate.

On the other hand, the Glu⁶⁰residue inTacTAL does not perform that protonation.

Tyrosine side chains are known to perform proton transfer reactions in other class I aldolases as in the case of the mammalian fructose-1,6-bis(phosphate) aldolase (FBPA). During the catalytic reaction of the rabbit muscle FBPA the C-terminal tyrosine residue (Tyr³⁶³) located on the flexible terminus acts together with Lys¹⁴⁶and Glu¹⁸⁷as the acid-base catalyst for the aldolytic cleavage of the substrate fructose 1,6-bis(phosphate) into the dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (St-Jean andSygusch, 2007). Interestingly, the deletion of this tyrosine residue by treatment of the protein with carboxypeptidase results in a decrease of the aldolase activity and in an increase of the transaldolase activity (Roseet al., 1965).

The relative orientation of the tyrosine residue in the active site ofEcFSA differs from that of the glutamate inTacTAL. While the transaldolase’s glutamate residue is oriented in same direction as the reactive lysine residue (towards the active site entrance), the aldolase’s tyrosine residue is oriented in the opposite direction (towards the reactive lysine). These two situations are common in different transaldolases and aldolases and are named as co-aligned and opposite-faced, respectively (Tittmann, 2014).

TacTAL was the first transaldolase in whose active site a reactive F6P-Schiffbase intermediate could be trappedin crystallo(Lehwess-Litzmannet al., 2011b). The C3 and C5 hydroxy groups of

OH

HN

HO

OH

OH

2-O3PO Lys86

O Asp₆

O Asn₂₈

H₂O Thr₁₁₀

O

O Glu60

Ser₁₃₀ Asn₁₀₈ Ser58

Arg₁₃₅

Arg169 Ser₁₆₇

Scheme 2.4: Coordination of the F6P-Schiff base intermediate in the active site of TacTAL. Adapted from Lehwess-Litzmann et al. (2011b)

the intermediate are coordinated by Asp⁶(figure 2.4). The C4 hydroxy group is coordinated by the Asn²⁸residue and the catalytic water molecule. The phosphate group is coordinated by the residues Arg¹³⁵, Arg¹⁶⁹and Ser¹⁶⁷.

The C1 hydroxy group exhibits two alternative conformations („up“ and „down“). This group in the „up“ conformation is coordinated by the residue Thr¹¹⁰ (figure 2.1.6 A) and the „down“

conformation is coordinated by the residue Ser¹³⁰(figure 2.1.6 B). Both interactions were analyzed in previous works (Lehwess-Litzmann, 2011; Sautner, 2012). TheTacTAL_T110V variant with interrupted „up“ coordination showed a greatly impaired transaldolase activity (Lehwess-Litzmann, 2011). Apart from the coordination of the C1 hydroxy group, this threonine coordinates the catalytic water molecule and is suggested to coordinate the C2 hydroxy group of the carbinolamine inter-mediate (Sch¨orkenet al., 2001). TheTacTAL_S130Avariant with interrupted „down“ coordination exhibits an impaired donor half-reaction activity (Sautner, 2012). However, crystallographic stud-ies on this variant showed that the C1 hydroxy group of the F6P-Schiffbase intermediate trapped in the active site adopts both alternative conformations despite the missing interaction partner (Ser¹³⁰) in the variant. In the case of the variant, the „down“ conformation is coordinated by the residue Asn¹⁰⁸, which is spatially close to the original interaction partner Ser¹³⁰. A corresponding variant of transaldolase fromEscherichia coli(EcTAL_S176A) showed highly impaired transaldolase activity (Schorken¨ et al., 2001).

F6P-Schiff

Figure 2.1.6: „Up“ and „down“ conformations of the C1 hydroxy group of the F6P-Schiff base intermediate in the active site ofTacTALwtco-crystallized with F6P. The selected active site residues (gray) are represented as sticks. The polypeptide backbone is shown as secondary structure (cyan). The catalytic water molecule is shown as the red sphere. The respective hydrogen bond interactions are represented as black dashes.A:The „up“ conformation of the intermediate (yellow) is coordinated by Thr¹¹⁰ (framed). B: The „down“ conformation of the intermediate (yellow) is coordinated by Ser¹³⁰(framed).