The quaternary structure of Thermus thermophilus aldehyde dehydrogenase is stabilized by an evolutionary distinct C-terminal arm extension

Abstract

Aldehyde dehydrogenases (ALDH) form a superfamily of dimeric or tetrameric enzymes that catalyze the oxidation of a broad range of aldehydes into their corresponding carboxylic acids with the concomitant reduction of the cofactor NAD(P) into NAD(P)H. Despite their varied polypeptide chain length and oligomerisation states, ALDHs possess a conserved architecture of three domains: the catalytic domain, NAD(P)⁺ binding domain, and the oligomerization domain. Here, we describe the structure and function of the ALDH from Thermus thermophilus (ALDH_Tt) which exhibits non-canonical features of both dimeric and tetrameric ALDH and a previously uncharacterized C-terminal arm extension forming novel interactions with the N-terminus in the quaternary structure. This unusual tail also interacts closely with the substrate entry tunnel in each monomer providing further mechanistic detail for the recent discovery of tail-mediated activity regulation in ALDH. However, due to the novel distal extension of the tail of ALDH_Tt and stabilizing termini-interactions, the current model of tail-mediated substrate access is not apparent in ALDH_Tt. The discovery of such a long tail in a deeply and early branching phylum such as Deinococcus-Thermus indicates that ALDH_Tt may be an ancestral or primordial metabolic model of study. This structure provides invaluable evidence of how metabolic regulation has evolved and provides a link to early enzyme regulatory adaptations.

Figure 1

Top. ALDH_Tt monomeric and tetrameric domain architecture in ribbon illustration. Monomer A consists of the catalytic domain (green), NAD(P)⁺ binding domain (blue), short linker loop (yellow), inter-domain linker (orange), oligomerisation domain (cyan) and the C-terminal tail (red). Bottom. Surface representation of the tetrameric assembly of ALDH_Tt showing intimate relationship between protomers. Coloring for monomer A’s domains is kept consistent to aid orientation. Monomers B, C and D are colored in purple, light brown, and pink respectively.

Figure 2

NADP⁺ binding and catalytic residues conformations in ALDH_Tt. (A) NADP⁺ binding highlighting hydrogen bonding. The cofactor and interacting residues are in stick representation, the FoFc omit map of NADP⁺ contoured at 3 sigma is shown as green mesh. (B) Close up view of Glu261 positioning in native (orange) and ALDH_Tt515 structures (Cyan). The catalytic cysteine 295 is shown for the native structure. The cofactor is shown to highlight the Glu261 movement induced by cofactor binding. (C) Superimposition of ALDH_TtNative and C_t-FDH structures (PDB code2O2P) showing similarities between the two structures in terms of Glutamic acid orientation and proximity to the catalytic cysteine which is present in double conformation.

Figure 3

Linker loop residues orientation. Structures superimposition showing linker loop residues orientations in native (Cyan) and NADP⁺ bound (Orange) structures. The catalytic cysteine is in the attacking conformation for both structures, while the glutamic acid is in the “In” conformation for the native structure or the intermediate conformation for the NADP-bound structure. The orientation of the residues from the linker loop Leu262 and Gly263 is shown. The hydrogen bound stabilizing the glutamic acid is shown in dashes.

Figure 4

Propanoic acid in the substrate entry channel. (A) Product binding in monomer A of the full length recombinant ALDH_Tt. The residues of the anchor loop (Cyan) are shown in stick and labeled. Potential hydrogen bonds between the propanoic acid (Green) and the protein residues are shown. (B) Product binding in monomer B, highlighting a different orientation of the carboxyl group. Glu261 orientation is shown only when the side chain was visible in the structure. The distance between the propanoic acid and the cysteine residue is shown in both structures.

Figure 5

Comparison of ALDH tail orientations with regard to the substrate entry tunnel and fulcrum points on the tails. (A) Superimposition of ALDH_Tt (Red), Human dimeric ALDH3 (PDB: 3SZA, magenta) and tetrameric ALDH7A1 in the closed and open conformations (PDB: 4ZUL, yellow; 4ZUK, light blue respectively). Highlights how all previously characterized tails fold at the crook or notch and rest in the interdomain cleft (domain coloring of Fig. 1 used) whilst ALDH_Tt is orientated across the tetramer interface and away from the other tails after the hook. (B) Close up of the substrate entry channel showing interaction between the C-terminal tail Gln510 of monomer B (brown) with Ala 465 of monomer A (Cyan) in ALDH_Tt. (C) Comparison of human ALDH7A1 and ALDH_Tt. Domain coloring is kept consistent with Fig. 1 for orientation purposes. The human dehydrogenase in the closed position can swing out away from the substrate entrance channel into the open conformation and fold into the interdomain cleft and against the catalytic domain due to its size whilst ALDH_Tt’s extended tail makes this impractical. (D) A magnified view of ALDH_Tt SEC is shown highlighting the positively charged K105 packing against the negatively charged hook of the tail and not allowing the tail to fold like those previously characterized. The crook is seen to be firmly over the SEC. NADP⁺ is depicted in stick within the tunnel.

Figure 6

Positioning of BOG in the native structure deep within the tetramer. (A) Surface representation of the tetramer in which monomer B has been removed for clarity purposes. The two BOG molecules (in stick representation) are deeply buried within the tetramer. Note that BOG molecules lie between the inter-domain linker and the oligomerization domain (Orange and cyan, respectively for monomer A). Coloring for all monomers is kept consistent with Fig. 1 to aid orientation. (B) Yellow dashed ellipse highlights the highly positive tunnel which exists between monomer interfaces.