Contribution of a C-Terminal Extension to the Substrate Affinity and Oligomeric Stability of Aldehyde Dehydrogenase from Thermus thermophilus HB27

Abstract

Aldehyde dehydrogenase enzymes (ALDHs) are widely studied for their roles in disease propagation and cell metabolism. Their use in biocatalysis applications, for the conversion of aldehydes to carboxylic acids, has also been recognized. Understanding the structural features and functions of both prokaryotic and eukaryotic ALDHs is key to uncovering novel applications of the enzyme and probing its role in disease propagation. The thermostable enzyme ALDHTt originating fromThermus thermophilus, strain HB27, possesses a unique extension of its C-terminus, which has been evolutionarily excluded from mesophilic counterparts and other thermophilic enzymes in the same genus. In this work, the thermophilic adaptation is studied by the expression and optimized purification of mutant ALDHTt-508, with a 22-amino acid truncation of the C-terminus. The mutant shows increased activity throughout production compared to native ALDHTt, indicating an opening of the active site upon C-terminus truncation and giving rationale into the evolutionary exclusion of the C-terminal extension from similar thermophilic and mesophilic ALDH proteins. Additionally, the C-terminus is shown to play a role in controlling substrate specificity of native ALDH, particularly in excluding catalysis of certain large and certain aromatic ortho-substituted aldehydes, as well as modulating the protein's pH tolerance by increasing surface charge. Dynamic light scattering and size-exclusion HPLC methods are used to show the role of the C-terminus in ALDHTt oligomeric stability at the cost of catalytic efficiency. Studying the aggregation rate of ALDHTt with and without a C-terminal extension leads to the conclusion that ALDHTt follows a monomolecular reaction aggregation mechanism.

Figure 1

Sequence alignment of the C-termini of thermophilic and mesophilic aldehyde dehydrogenases. Alignment of sequences was performed using Clustal Omega and displayed using GeneDoc software. Purple areas represent regions of highly conserved homology. The blue dashed box displays the characteristic 30 amino acid tail of ALDHTt.

Figure 2

Relationship between the substrate access tunnel and C-terminal tail in ALDHTt-native (A) and the ALDHTt-native tetrameric structure as a ribbon model (B). All four monomers of the structure are labeled in the surface model of ALDHTt-native in (C). The monomers are labeled as follows; A, blue; B, pink; C, green; and D, cyan. (D) Repulsion forces between Lys105 (shown in magenta) and the “hook” of the tail, causing the tail to extend toward the N-terminus of the relative monomer. Panel (A) was adapted from ref (15). Available under a CC-BY 4.0 license. Copyright 2018 Kevin Hayes et al.

Figure 3

SDS Page (A) and Western blot gels (B) from the production of ALDHTt-508. (Lane 0) molecular weight marker (PageRuler Prestained Protein ladder, ThermoFisher), (lane 1) cell lysate, (lane 2) nickel affinity eluted waste, (lane 3) elution at 200 mM Imidazole, and (lane 4) elution at 500 mM imidazole.

Figure 4

Temperature (A) and pH (B) profiles of ALDHTt-508 using an NAD+ coupled assay and hexanal as the substrate.

Figure 5

Screening of ALDHTt-508 aldehyde specificity at 25 °C (A) and 50 °C (B). The enzyme has a broad substrate specificity toward a range of aldehydes, particularly at higher temperatures.

Figure 6

Effect of the loss of the C-terminal extension on the aggregation profiles of ALDHTt-native and ALDHTt-508 measured by dynamic light scattering (DLS). The intensity and hydrodynamic diameter data was collected using the Zetasizer Nano-ZSP DLS and normalized using Prism GraphPad (Ver 10.0.1) and fit to a plateau, followed by a one-phase association equation. The mean of three independent experiments is presented.

Figure 7

Aggregation kinetics of ALDHTt-native (green) and ALDHTt-508 (red) at temperatures of 65 (A) and 80 °C (B), as measured by dynamic light scattering (DLS). The intensity of diffraction is presented as a function of time. The samples were prepared at 800 μg/mL in 10 mM KPO₄ buffer and filter sterilized. The experimental data points (solid lines) show the mean of at least two independent experiments. The result of fitting (eq 3) is shown by dotted lines. I_lim is defined as the final point of aggregation that can be detected by DLS.

Figure 8

Dependence of hydrodynamic diameter on the intensity of scattered light of 508 ALDHTt-native (A) and ALDHTt-508 (B). The vertical dotted line corresponds to the value 509 of D_H at room temperature.

Figure 9

Electrostatics of ALDHTt (dimer) and its truncated mutant, ALDHTt-508. The figure shows the dual polarity of the C-terminal tail (black dashed circle) and the surface charges exposed upon its removal. All ligands from ALDHTt-native and ALDHTt-508 crystallographic data have been removed for clarity (PDB: 6FJX and 6FKV, respectively). Figures were modeled using the Adaptive Poisson–Boltzmann Solver (APBS) available on PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC).