Structural Analysis of an Evolved Transketolase Reveals Divergent Binding Modes

Affaticati, Pierre E.; Dai, S.; Payongsri, Panwajee; Hailes, Helen C.; Tittmann, Kai; Dalby, Paul A.

doi:10.1038/srep35716

Cited by 18 publications

(29 citation statements)

References 37 publications

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“…The high number of inhibitory subclusters in both 385 p AMF and 385Y was consistent with the high cooperativity of inhibition, and high theoretical number of inhibitory binding orientations ( n i ) observed experimentally. The two catalytically productive subclusters of each variant were split between the two binding pockets that had been previously identified [15] (Fig. 9A).…”

Section: Resultsmentioning

confidence: 99%

Fine‐tuning the activity and stability of an evolved enzyme active‐site through noncanonical amino‐acids

Wilkinson

Dalby

2020

The FEBS Journal

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Site-specific saturation mutagenesis within enzyme active sites can radically alter reaction specificity, though often with a trade-off in stability. Extending saturation mutagenesis with a range of noncanonical amino acids (ncAA) potentially increases the ability to improve activity and stability simultaneously. Previously, an Escherichia coli transketolase variant (S385Y/D469T/R520Q) was evolved to accept aromatic aldehydes not converted by wild-type. The aromatic residue Y385 was critical to the new acceptor substrate binding, and so was explored here beyond the natural aromatic residues, to probe side chain structure and electronics effects on enzyme function and stability. A series of five variants introduced decreasing aromatic ring electron density at position 385 in the order para-aminophenylalanine (pAMF), tyrosine (Y), phenylalanine (F), para-cyanophenylalanine (pCNF) and para-nitrophenylalanine (pNTF), and simultaneously modified the hydrogen-bonding potential of the aromatic substituent from accepting to donating. The fine-tuning of residue 385 yielded variants with a 43-fold increase in specific activity for 50 mM 3-HBA and 100% increased k cat (pCNF), 290% improvement in K m (pNTF), 240% improvement in k cat /K m (pAMF) and decreased substrate inhibition relative to Y. Structural modelling suggested switching of the ring-substituted functional group, from donating to accepting, stabilised a helix-turn (D259-H261) through an intersubunit H-bond with G262, to give a 7.8°C increase in the thermal transition mid-point, T m , and improved packing of pAMF. This is one of the first examples in which both catalytic activity and stability are simultaneously improved via site-specific ncAA incorporation into an enzyme active site, and further demonstrates the benefits of expanding designer libraries to include ncAAs.

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Section: Resultsmentioning

confidence: 99%

Fine‐tuning the activity and stability of an evolved enzyme active‐site through noncanonical amino‐acids

Wilkinson

Dalby

2020

The FEBS Journal

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“…Besides, an isoleucine found in Ec TK and Sc TK in standard position 189 is replaced by L191 in Gst TK . Aspartate in standard position 469 plays a role in substrate binding, activity enhancement and stereoselectivity, is conserved among the three TKs mentioned above and found in 59 % of TK sequences. For further details and references see SI Table S2.…”

Section: Resultsmentioning

confidence: 99%

Towards a Mechanistic Understanding of Factors Controlling the Stereoselectivity of Transketolase

et al. 2018

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A structural model for thiamine‐diphosphate (ThDP)‐dependent transketolase (TK) was developed to analyse the effect of amino acid exchanges on the stereoselectivity of this synthetically important class of enzymes. In this study the carboligation of 3‐hydroxypyruvate as a donor and propanal, as well as pentanal, was studied. Based on literature data and additional mutagenesis studies using E. coli TK, a four‐state model was developed to explain the stereoselectivity of TKs by the relative orientation of donor and acceptor substrates in the active site prior to C−C‐bond formation. To enable a functional comparison of relevant amino acids of TKs from different species, a standard numbering scheme was developed. Using this concept, H26, H261, and F434 were identified as the key residues which mediate stereoselectivity, where two main factors influenced the arrangement of ThDP‐bound donor and acceptor prior to carboligation: the relative orientation of the substrate side chains and the orientation of the acceptor carbonyl group towards the donor hydroxy group. This model provides a first framework to understand the structure‐function relationships of TKs with respect to their stereoselectivity.

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“…In aldolases, the population of a non-productive binding pose can increase at low pH 36 . Therefore, knowing the determinants of non-productive binding poses is crucial for rational protein design 37 , and improving the ratio between productive and non-productive binding might be the underlying principle of increasing catalytic activity upon directed evolution 38 . Blocking of non-productive binding sites could explain the activating effect of effector molecules like warfarin for CYP2C9 32 or carboxylic acid for oleate hydratase 39,40 .…”

Section: Discussionmentioning

confidence: 99%

Modelling of substrate access and substrate binding to cephalosporin acylases

Ferrario

Fischer

Zhu

et al. 2019

Sci Rep

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Semisynthetic cephalosporins are widely used antibiotics currently produced by different chemical steps under harsh conditions, which results in a considerable amount of toxic waste. Biocatalytic synthesis by the cephalosporin acylase from Pseudomonas sp . strain N176 is a promising alternative. Despite intensive engineering of the enzyme, the catalytic activity is still too low for a commercially viable process. To identify the bottlenecks which limit the success of protein engineering efforts, a series of MD simulations was performed to study for two acylase variants (WT, M6) the access of the substrate cephalosporin C from the bulk to the active site and the stability of the enzyme-substrate complex. In both variants, cephalosporin C was binding to a non-productive substrate binding site (E86α, S369β, S460β) at the entrance to the binding pocket, preventing substrate access. A second non-productive binding site (G372β, W376β, L457β) was identified within the binding pocket, which competes with the active site for substrate binding. Noteworthy, substrate binding to the protein surface followed a Langmuir model resulting in binding constants K = 7.4 and 9.2 mM for WT and M6, respectively, which were similar to the experimentally determined Michaelis constants K M = 11.0 and 8.1 mM, respectively.

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Structural Analysis of an Evolved Transketolase Reveals Divergent Binding Modes

Cited by 18 publications

References 37 publications

Fine‐tuning the activity and stability of an evolved enzyme active‐site through noncanonical amino‐acids

Fine‐tuning the activity and stability of an evolved enzyme active‐site through noncanonical amino‐acids

Towards a Mechanistic Understanding of Factors Controlling the Stereoselectivity of Transketolase

Modelling of substrate access and substrate binding to cephalosporin acylases

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