Structural analysis of pathogenic mutations targeting Glu427 of ALDH7A1, the hot spot residue of pyridoxine-dependent epilepsy

Abstract

Certain loss-of-function mutations in the gene encoding the lysine catabolic enzyme aldehyde dehydrogenase 7A1 (ALDH7A1) cause pyridoxine-dependent epilepsy (PDE). Missense mutations of Glu427, especially Glu427Gln, account for ~30% of the mutated alleles in PDE patients, and thus Glu427 has been referred to as a mutation hot spot of PDE. Glu427 is invariant in the ALDH superfamily and forms ionic hydrogen bonds with the nicotinamide ribose of the NAD⁺ cofactor. Here we report the first crystal structures of ALDH7A1 containing pathogenic mutations targeting Glu427. The mutant enzymes E427Q, Glu427Asp, and Glu427Gly were expressed in Escherichia coli and purified. The recombinant enzymes displayed negligible catalytic activity compared to the wild-type enzyme. The crystal structures of the mutant enzymes complexed with NAD⁺ were determined to understand how the mutations impact NAD⁺ binding. In the E427Q and E427G structures, the nicotinamide mononucleotide is highly flexible and lacks a defined binding pose. In E427D, the bound NAD⁺ adopts a “retracted” conformation in which the nicotinamide ring is too far from the catalytic Cys residue for hydride transfer.Thus, the structures revealed a shared mechanism for loss of function: none of the variants are able to stabilise the nicotinamide of NAD⁺ in the pose required for catalysis. We also show that these mutations reduce the amount of active tetrameric ALDH7A1 at the concentration of NAD⁺ tested. Altogether, our results provide the three-dimensional molecular structural basis of the most common pathogenic variants of PDE and implicate strong (ionic) hydrogen bonds in the aetiology of a human disease.

1 |. INTRODUCTION

Pyridoxine-dependent epilepsy (PDE) is an epileptic encephalopathy characterised by seizures, usually occurring in the first hours of life, which are unresponsive to standard antiepileptic drugs, but can be controlled by pharmacologic doses of pyridoxine.¹ The most common cause of PDE is autosomal recessive inheritance of certain mutations in the gene encoding the enzyme α-aminoadipate semialdehyde dehydrogenase, also known as ALDH7A1 or antiquitin. ALDH7A1 is part of lysine catabolism and catalyses the NAD⁺-dependent oxidation of α-aminoadipate semialdehyde (AASAL) to α-aminoadipate (AA). The mutational spectrum of PDE spans over 113 different mutations within the 18 exons of the ALDH7A1 gene, including approximately 60 missense mutations.^1–4 The estimated carrier frequency of ALDH7A1 mutations is 1:127, and the estimated incidence of ALDH7A1 deficiency is 1:64 352 births.¹ The biochemical basis of PDE is a decrease of the ubiquitous enzyme cofactor pyridoxal 5’-phosphate (PLP). The cellular pool of PLP is depleted via the irreversible reaction of PLP with Δ¹-piperideine-6-carboxylic acid (P6C), the cyclised form of AASAL.⁵ Treatment with pyridoxine addresses the PLP deficiency and typically provides adequate seizure control, yet 75% of individuals with PDE have intellectual disability and developmental delay.^2,3

The missense mutation of Glu427 to glutamine (Glu427Gln) is the most commonly reported pathogenic variant in the PDE literature,¹ and this mutation is estimated to be present in 30% of PDE patients with European ancestry.^5–7 In addition to the Gln mutation at residue 427, mutations to Asp (Glu427Asp) and Gly (Glu427Gly) were reported in a cohort of 18 North American patients with PDE.⁸ Because of its high frequency of mutation in PDE patients, Glu427 has been referred to as a mutational hotspot of the disease.⁸

Glu427 plays a key role in binding the cofactor NAD⁺. Crystal structures of ALDHs, including ALDH7A1, show that Glu427 hydrogen bonds to the hydroxyl groups of the NAD⁺ nicotinamide ribose (Figure 1A). Presumably, these interactions help stabilise the extended conformation of NAD⁺, and ensure that the nicotinamide ring is close to the catalytic cysteine (Cys330) and positioned optimally for the hydride transfer step of the catalytic mechanism. The importance of these hydrogen bonds is highlighted by the fact that Glu427 is one of only a few invariant residues conserved throughout the entire ALDH superfamily.⁹

FIGURE 1.

Structural context of NAD⁺ binding and steady-state kinetic data for Glu427 variants. A, Conformation and interactions of NAD⁺ bound to ALDH7A1 in the active conformation (PDB 6O4C). For reference, NAD⁺ and protein are coloured sand and grey, respectively. Steady-state kinetic data for (B) E427Q, (C) E427D, and (D) E427G. In each panel, the black symbols are data for the wild-type enzyme, and the black curve is the fit to the Michaelis-Menten equation. The circles, squares, and triangles represent different replicate experiments performed at the same substrate concentration. Three trials were performed for each enzyme. The kinetic constants for the wild-type enzyme are k_cat = 0.38 ± 0.02 seconds⁻¹ and K_m = 517 ± 86 μM

To better understand the molecular basis of the most common pathogenic variants of PDE, we have determined the catalytic properties, crystal structures, and oligomeric states of the ALDH7A1 pathogenic mutant variants E427Q, E427D, and E427G. None of the variants exhibit measurable catalytic activity, consistent with a role in PDE pathogenesis. The crystal structures show that the catalytic defect results from NAD⁺ adopting non-native conformations, which are incompatible with catalysis. Also, the mutations reduce the amount of active tetrameric ALDH7A1 under the conditions tested. These results provide a molecular-structural explanation of the most common pathogenic variants of PDE.

2 |. RESULTS

2.1 |. The mutations E427Q, E427D, and E427G compromise catalytic activity

The purified recombinant mutant variants E427Q, E427D, and E427G were tested for catalytic activity using steady-state assays that monitor the production of NADH. As a control, the activity of the wild-type enzyme was assayed as a function of AASAL concentration with NAD⁺ fixed at a saturating concentration of 2.5 mM. The enzyme displays Michaelis-Menten behaviour (Figure 1B–D), characterised by kinetic constants of k_cat = 0.38 ± 0.02 seconds⁻¹ and K_m for AASAL of 517 ± 86 μM. The resulting catalytic efficiency (k_cat/K_m) of 735 seconds⁻¹ M⁻¹ is within a factor of 2 to 6 of the values reported previously.^10–12 In contrast, none of the mutant variants displayed detectable activity across the entire range of AASAL concentration (Figure 1B–D). The results for E427Q and E427G agree with previous studies reporting an absence of catalytic activity in crude extracts of cells expressing these variants.^5,13 To our knowledge, ours is the first report of E427D being inactive.

2.2 |. The nicotinamide mononucleotide of NAD⁺ bound to E427Q and E427G is highly flexible and lacks a defined pose

The crystal structure of E427Q complexed with NAD⁺ was determined in space group C2 at 2.06 Å resolution from a crystal grown in the presence of 15 mM NAD⁺.The asymmetric unit contains eight protein chains arranged in two tetramers. The tetramer observed in the crystal has been shown to be the active form of ALDH7A1.¹⁴ We note this is the same crystal form that has been used to solve structures of wild-type ALDH7A1^15,16 and several PDE mutant variants.¹¹

The electron density was sufficient to model the loop containing Gln427 (“427-loop”) in all eight chains of the asymmetric unit (Figure 2A). The conformation of the 427-loop of E427Q is very similar to that of the wild-type enzyme (Figure 2B). Therefore, the defect in catalysis is not due to a major structural perturbation of the 427-loop.

FIGURE 2.

The impact of E427Q/D/G mutations on the structure of ALDH7A1. A, Electron density for NAD⁺ and the 427-loop of E427Q. The cage represents polder omit maps (3σ). B, Superposition of E427Q-NAD⁺ (sand) with the wild-type enzyme (white, PDB code 2J6L). The dashed lines represent hydrogen bonds in the wild-type enzyme between Glu427 and NAD⁺. C, Electron density for NAD⁺ and the 427-loop of E427G. The cage represents polder omit maps (3σ). D, Superposition of E427G-NAD⁺ (sand) with the wild-type enzyme (white, PDB code 2J6L). The arrow denotes the 3-Å shift of residue 427 caused by the mutation. E, Electron density for NAD⁺ and the 427-loop of E427D. The cage represents polder omit maps (3σ). The asterisk denotes the C4 atom of the nicotinamide, which is the hydride acceptor of the ALDH reaction. F, Superposition of E427D-NAD⁺ (sand) with the wild-type enzyme (white, PDB code 2J6L). The curved arrow denotes the 7-Å shift in the nicotinamide caused by the mutation

Strong electron density was observed for only the ADP portion of NAD⁺, suggesting the nicotinamide mononucleotide (NMN) group of the bound cofactor is highly flexible (Figure 2A). This was true for all eight protein chains in the asymmetric unit. We note that the NMN is also disordered in wild-type ALDH7A1 structures when NAD⁺ is included in the crystallisation at 5 mM (eg, see PDB code 4ZUK).¹⁵ However, the cofactor is fully resolved by strong electron density when included in the crystallisation in the higher concentration range of 11 to 15 mM (PDB codes 2J6L, 6O4B-C, 6O4E, 6O4H).^11,16 Because 15 mM NAD⁺ was used for crystallisation of E427Q, the lack of electron density for the NMN is meaningful. The apparent partial binding of the cofactor under these conditions is consistent with the possibility of the mutation significantly increasing the K_m for NAD⁺ compared to wild-type.

The resolved ADP fragment of NAD⁺ bound to E427Q exhibits an atypical conformation. Although the adenosine of NAD⁺ binds the canonical location, the pyrophosphate group has rotated into an unusual “out” conformation in six of the eight chains (chains A-F) in the asymmetric unit (Figure 2A,B). In chains A and E, density for only the “out” conformation is observed, while strong density for both the “out” and a more typical (“in”) conformation is present in chains B-D,F (eg, Figure 2A). In the “out” conformation, the NMN appears to be sampling the solvent region outside of the active site (Figure 2B). We note the “out” conformation has not been observed in any other ALDH7A1 structure.

The structure of E427G-NAD⁺ (2.15 Å resolution) tells a similar story. As in E427Q-NAD⁺, the NMN of NAD⁺ bound to E427G is disordered, and there is electron density evidence suggesting an “out” conformation in some of the chains (Figure 2C). We note that electron density for the 427-loop is weaker than in the other mutant variants, particularly at residue 427 (Figure 2C). This is likely due to the increased flexibility introduced by the mutation of Glu to Gly. Nevertheless, the density was sufficient for modelling the backbone of the loop in all eight chains (Figure 2C). The model shows that residue 427 has shifted by 2 to 3 Å away from binding site for the nicotinamide ribose (Figure 2D). Thus, unlike E427Q, the mutation to Gly apparently perturbs the local protein conformation.

2.3 |. NAD⁺ binds to E427D in a retracted, inactive pose

The crystal structure of E427D complexed with NAD⁺ was determined in space group P2₁ at 2.06 Å resolution. We note this crystal form is new for ALDH7A1. The electron density is strong for the entire NAD⁺ cofactor in all four chains in the asymmetric unit. Likewise, the map guided the modelling of the 427-loop in all four chains. The density in chain A provides the clearest picture of the 427-loop conformation, so we focus our analysis on this chain (Figure 2E).

The NAD⁺ adopts an inactive conformation. While the adenosine is anchored in the canonical binding site, the NMN has withdrawn from active site by 4 to 7 Å (Figure 2F). The retracted conformation is stabilised by several interactions not found in the wild-type enzymecofactor complex: the carboxamide of the nicotinamide hydrogen bonds with Asp427 and Gly299, the nicotinamide ribose hydrogen bonds with Asp427, and the nicotinamide ring stacks against Phe429.

The retracted conformation is incompatible with catalysis. The hydride transfer step of the ALDH mechanism requires proximity of the nicotinamide to catalytic Cys330. In the wild-type enzyme, the hydride acceptor atom of the nicotinamide (C4) is 3.0 Å from the S atom of Cys330. The corresponding distance in E427D is 4.4 Å, which is unsuitable for hydride transfer (Figure 2F).

A structure of E427D complexed with the product AA was also determined (1.75 Å resolution) to understand how this mutation affects recognition of the aldehyde substrate. The pose and interactions of AA bound to E427D are identical to those of the wild-type enzyme (Figure S1). This result supports the hypothesis that retraction of NAD⁺ from the catalytic Cys is the main cause of the catalytic defect of E427D.

2.4 |. Mutation of Glu427 reduces the amount of tetrameric ALDH7A1

We previously showed that the active form of ALDH7A1 is a tetramer, and the binding of NAD⁺ promotes tetramer assembly.¹⁴ Because the mutant variants fail to bind NAD⁺ in the active conformation, we tested the ability of these enzymes to form the tetramer. Small-angle X-ray scattering (SAXS) was used to investigate the oligomeric structure.¹⁷

As previously observed, the addition of NAD⁺ (10 mM) to wild-type ALDH7A1 results in complete tetramer formation as indicated by the excellent agreement (χ² < 1) between the experimental SAXS curve and the theoretical SAXS curve calculated from the crystallographic tetramer model (Table 1, Figure S2). This result indicates that the binding of NAD⁺ shifts the dimer-tetramer equilibrium of the wild-type enzyme overwhelmingly to the tetramer, in agreement with our previous study.¹⁴

TABLE 1.

Small-angle X-ray scattering analysis

	ALDH7A1			E427Q			E427D			E427G
	1.2 mg/ml	2.3 mg/ml	4.7 mg/ml	1.1 mg/ml	2.1 mg/ml	4.3 mg/ml	1.4 mg/ml	2.9 mg/ml	5.7 mg/ml	1.6 mg/ml	3.2 mg/ml	6.5 mg/ml
Guinier analysis
Rg(Å)	38.3 ± 0.2	38.3 ± 0.2	38.3 ± 0.2	37.4 ± 0.2	37.8 ± 0.2	37.6 ± 0.2	36.9 ± 0.2	38.2 ± 0.2	38.5 ± 0.2	37.8 ± 0.2	37.4 ± 0.2	37.6 ± 0.2
qmin (Å−1)	0.01087	0.01087	0.01087	0.01087	0.01087	0.01087	0.01087	0.01087	0.01087	0.01087	0.01087	0.01087
qRg range	0.42–1.29	0.42–1.29	0.42–1.29	0.41–1.30	0.41–1.29	0.41–1.29	0.40–1.30	0.41–1.29	0.42–1.30	0.41–1.29	0.41–1.28	0.41–1.29
R-Squared	0.937	0.991	0.988	0.969	0.981	0.994	0.957	0.996	0.998	0.98	0.988	0.998
I(0)	16.06 ± 0.07	31.1 ± 0.1	65.2 ± 0.2	15.29 ± 0.06	31.5 ± 0.1	61.0 ± 0.2	22.26 ± 0.08	47.7 ± 0.2	95.5 ± 0.3	22.83 ± 0.09	50.2 ± 0.2	103.8 ± 0.3
P(r) analysis
Rg (Å)	37.5 ± 0.2	37.4 ±0.1	37.5 ± 0.8	36.6 ± 0.1	36.6 ± 0.1	36.6 ± 0.1	37.8 ± 0.1	37.9 ± 0.8	37.7 ± 0.8	37.2 ± 0.2	37.2 ± 0.2	37.6 ± 0.4
Dmax (Å)	115	107	107	102	108	106	113	114	106	113	105	109
q-Range (Å−1)	0.0125–0.2082	0.0120–0.2088	0.0137–0.2082	0.0131–0.2160	0.0142–0.2121	0.0142–0.2121	0.0125–0.2099	0.0114–0.2093	0.0125–0.2076	0.0125–0.2138	0.0131–0.2132	0.0120–0.2127
Total quality estimate	0.92	0.95	0.93	0.94	0.87	0.92	0.96	0.94	0.82	0.91	0.79	0.76
Porod volume (Å3)	350 000	326 000	315 000	237 000	237 000	255 000	256 000	259 000	272 000	291 000	251 000	271 000
SAXS Mr (kDa)
MoWa	211 000	221 000	230 000	175 000	180 000	192 000	158 000	185 000	206 000	196 000	186 000	205 000
Vcb	191 300	190 000	193 000	154 000	159 000	168 000	155 000	170 000	180 000	170 000	167 000	178 000
Model fitting
Dimer χ2	17	50	110	16	26	62	21	52	120	26	49	140
Tetramer χ2	0.52	0.39	0.65	1.0	1.1	1.9	1.4	1.1	2.3	1.0	1.2	2
Dimer: tetramer (χ2)	N/Dc	N/Dc	N/Dc	40:60 (0.83)	29:71 (0.72)	26:74(1.1)	18:82 (1.4)	14:86 (1.0)	9:91 (2.3)	33:67 (0.74)	22:78 (0.82)	13:87 (1.6)
SASBDB code	SASDGH4	SASDGJ4	SASDGK4	SASDGL4	SASDGM4	SASDGN4	SASDGP4	SASDGQ4	SASDGR4	SASDGS4	SASDGT4	SASDGU4

^aCalculated using the SAXS MoW method¹⁸ as implemented in PRIMUS.¹⁹

^bCalculated using the volume of correlation method²⁰ as implemented in PRIMUS.¹⁹

^cUsing a dimer-tetramer ensemble did not improve the fit compared to the tetramer-only model.

All three ALDH7A1 Glu427 mutant variants were tested under the same experimental conditions (10 mM NAD⁺) and similar protein concentration ranges and compared to this wild-type result. Interestingly, all three mutant variants are compromised in tetramer formation. In each case, an ensemble model consisting of both the dimer and the tetramer structures provided better fits to the experimental data than the tetramer alone. Ensemble analysis with MultiFoXS shows that the E427Q, E427D, and E427G samples contain 26% to 40%, 9% to 18%, and 13% to 33% dimer, respectively (Table 1, Figure S2).

Two other metrics were also utilised to reveal discrepancies in the oligomeric state of wild-type vs mutant ALDH7A1: experimentally determined molecular mass (M_r) from SAXS and Porod volume (V_P). Wild-type samples showed a SAXS M_r of 211 to 230 kDa (Table 1), which is within 5% of the theoretical M_r of 222 kDa. In contrast, the E427Q samples showed a SAXS M_r of 175 to 192 kDa (Table 1). Note that at the highest concentration tested, the M_r of E427Q is 13% lower than that of the tetramer. Similarly, the SAXS M_r values of E427D and E427G are consistently lower (7%−29%) than expected for a predominantly tetrameric solution (Table 1). Further, the range of V_P reveals an overall decrease in average V_P of 26%, 21%, and 18% for E427Q, E427D, and E427G compared to wild-type (Table 1). Because of the relationship of V_P to approximate M_r,¹⁷ these data are also consistent with decreased M_r in the mutants compared to wild-type. Overall, these data are consistent with a decreased average in solution M_r for the Glu427 variants as compared to wild-type, suggesting the mutations perturbed the dimer-tetramer equilibrium.

3 |. DISCUSSION

The three ALDH7A1 Glu427 variants studied display negligible catalytic activity, consistent with clinical studies implicating them in the pathology of PDE.^1,5–8 We note that PDE patients who are biallelic for E427Q generally experience relatively early disease onset, which is consistent with the lack of activity of this mutant.⁷

The impact of mutating Glu427 has also been studied in ALDH2. Mutation of Glu427 of ALDH2 to Gln or Asp decreases catalytic efficiency by factors of 40 and 6, respectively.^21,22 We found that these mutations have a much more profound effect on the catalytic activity of ALDH7A1. A possible explanation for this difference is that the NAD⁺ bound to ALDH2 is stabilised by additional interactions not found in ALDH7A1. Trp168 of ALDH2 (Phe194 in ALDH7A1) forms two hydrogen bonds with the pyrophosphate of NAD⁺ (Figure S3).²³ These extra interactions may partially compensate for the loss of Glu427 in the ALDH2 variants.

Because Glu427 is invariant in the ALDH superfamily and participates directly in binding NAD⁺, it is perhaps not surprising that mutations of Glu427 of ALDH7A1 are pathogenic. However, from a physicochemical perspective, one might consider the mutation E427Q to be conservative. After all, glutamine is isostructural with glutamate, and glutamine has the potential to hydrogen bond to the nicotinamide ribose hydroxyl groups. The fact that glutamine is never observed at residue 427 suggests that the negative charge of glutamate is essential for optimal catalytic function.

The essentiality of Glu427 is likely due to its ability to form strong hydrogen bonds. In his seminal book on hydrogen bonding, George Jeffrey described the three categories of hydrogen bonds: strong, moderate, and weak.²⁴ Those between Glu427 and the hydroxyls of NMN are “strong” by virtue of the negative charge of glutamate. This type of hydrogen bond is also known as “ionic hydrogen bonds”. In contrast, glutamine can only form moderate hydrogen bonds with a hydroxyl group. Our results show that, apparently, moderate hydrogen bonds are not sufficient in energy to keep the NMN group in the proper position for catalysis, and thus the recognition of NAD⁺ by ALDH7A1 depends critically on the ability of Glu427 to form strong hydrogen bonds with the cofactor. We may conclude that the aetiology of this human disease has roots in the fundamental principles of physical chemistry.

Strong hydrogen bonds between residue 427 and the NMN may help promote the unfurling of NAD⁺ during binding. The predominant form of NAD⁺ in solution is a compact folded conformation in which the nicotinamide and adenine rings stack together with an inter-ring distance of 5.2 Å.²⁵ In contrast, NAD⁺ bound to ALDH7A1 (and other Rossmann fold enzymes²⁶) is highly extended, with an inter-ring distance of ~13 Å. Therefore, the enzyme must supply sufficient interactions to stabilise the extended conformation over the folded form. We observed that NAD⁺ adopts well-defined, extended conformations when bound to either the wild-type enzyme or E427D, and both enzymes form strong hydrogen bonds with the NMN. In contrast, NAD⁺ exhibits disorder in E427Q and E427G, which are incapable of forming strong hydrogen bonds. The “out” conformation of NAD⁺ observed in these variants perhaps suggests that the bound cofactor is sampling folded conformations. Thus, strong hydrogen bonds appear to be critical for promoting the unfolding of NAD⁺ during the process of binding to ALDH7A1.

4 |. CONCLUSION

Our results provide the three-dimensional molecular structural basis of the most common pathogenic variants of PDE. The lack of catalytic activity of E427Q, E427D, and E427G suggests that ALDH7A1 is very sensitive to mutations at Glu427. This is consistent with the universal conservation of this residue in the ALDH superfamily. In E427Q and E427G, the bound cofactor is highly flexible and lacks a defined conformation for the NMN group. In E427D, the cofactor adopts a novel inactive pose in which the nicotinamide ring is retracted from the active site. Thus, the structures revealed a shared mechanism for loss of function: none of the variants are able to stabilise the nicotinamide group of NAD⁺ in the pose required for catalysis.

Here we uncovered a third distinct molecular mechanism by which genetic mutations in the ALDH7A1 gene impair ALDH7A1 enzymatic function. These results build upon previous studies of mutations targeting the oligomer interfaces of ALDH7A1²⁷ and a set of mutations affecting residues in the AASAL binding site.¹¹ The interface mutations abrogated catalytic function by impairing formation of the active tetramer. In contrast, mutations in the AASAL site resulted in subtle perturbations of the structure and dynamics of the aldehyde substrate site, without affecting cofactor binding. The Glu427 variants revealed a third mechanism involving a defect in NAD⁺ binding, accompanied by a reduction in the amount of active tetramer formed.

5 |. MATERIALS AND METHODS

5.1 |. Protein expression and purification

Synthetic genes encoding E427Q, E427D, and E427G were generated using the Agilent QuikChange Lightning site-directed mutagenesis kit using the primers listed in Table S1. The mutations were verified by Sanger sequencing. Wild-type ALDH7A1 and the mutant variants were expressed in E. coli and purified as described previously.^11,27

5.2 |. Kinetic assays

ALDH7A1 enzymatic activity was measured by monitoring NADH production at a wavelength of 340 nm using an Epoch 2 Microplate Reader. The reaction assay buffer contained 50 mM pyrophosphate buffer (pH 8.0). Enzyme stock solutions were prepared by diluting to the desired concentration with 50 mM pyrophosphate buffer at pH 8.0 supplemented with 2.5 mM NAD⁺. AASAL was used as the variable substrate (16–2000 μM) with NAD⁺ fixed at 2.5 mM. AASAL was synthesised and quantitated as previously reported.¹⁰ The assays contained a final enzyme concentration of 0.07 μM wild-type and 5 μM E427Q/D/G. Triplicate data sets were collected, and kinetic constants were obtained by fitting the initial rate data to the Michaelis-Menten equation globally using Origin 2019.

5.3 |. Protein crystallisation

Crystallisation trials were performed at 20°C using sitting drop vapour diffusion. Wild-type ALDH7A1 crystals were prepared as described previously¹¹ and crushed to make a microseed stock to aid crystallisation of the mutant variants.

Crystals of E427Q and E427D complexed with NAD⁺ were grown by co-crystallisation using 5 mg/ml enzyme and 15 mM NAD⁺. The reservoir contained 0.2 M MgCl₂, 21% (w/v) PEG 3350, and 0.1 M Bis-Tris (at pH 6.2 for E427Q and pH 5.7 for E427D). Crystallisation trials (with microseeding) were set up using an Oryx8 robot (Douglas Instruments). The crystals were prepared for low temperature data collection by soaking in a cryobuffer consisting of the reservoir supplemented with 18% (v/v) ethylene glycol and 5 mM NAD⁺, followed by flash-cooling in liquid nitrogen.

Crystals of E427D complexed with AA were grown in 0.2 M MgCl₂, 0.1 M Bis-Tris pH 5.4, and 25% (w/v) PEG 3350. Prior to crystallisation, the enzyme (6 mg/ml) was incubated overnight with 80 mM AA. Microseeding with wild-type crystals was employed. The cryoprotectant consisted of the reservoir supplemented with 18% (v/v) ethylene glycol.

Crystals of E427G complexed with NAD⁺ were grown by co-crystallisation using 5 mg/ml enzyme and 5 mM NAD⁺. Wild-type crystals were used for microseed matrix-screening²⁸ using Hampton Index screen. Drops were set using an Oryx8 robot (Douglas Instruments). Data collection quality crystals grew in Hampton Index condition 71 (0.2 M NaCl, 0.1 M Bis-Tris pH 6.5, and 25% (w/v) PEG 3350). The cryoprotectant consisted of the reservoir supplemented with 18% (v/v) ethylene glycol.

X-ray diffraction data sets were collected at ALS beamline 4.2.2 using a Taurus-1 CMOS detector in shutterless mode. Each data set consisted of 900 images covering a rotation range of 180°with a total exposure time of 360 seconds. In some cases, two 180°scans, optimised to collect high and low resolution, were collected from the same crystal. The data sets were integrated and scaled with XDS.²⁹ Merging of low and high resolution scans was done with XSCALE.³⁰ Intensities were converted to amplitudes with AIMLESS.³¹ Data processing statistics are listed in Table S2. We note that the deposited structures use the historic numbering of ALDH7A1 to be consistent with other ALDH7A1 structures in the PDB. The two numbering schemes differ by 28 residues, and as such, Glu427 is Glu399 in the deposited structures.

Except for E427D-NAD⁺, the space group is C2, and the asymmetric unit contains eight protein chains arranged in two tetramers. This is the same crystal form that was used previously to determine structures of wild-type ALDH7A1^15,16 and several mutant variants.¹¹ Crystallisation trials of E427D-NAD⁺ generated a new ALDH7A1 crystal form with space group P2₁ and the following unit cell dimensions: a = 82 Å, b = 128 Å, c = 88 Å, and β = 101°. The asymmetric unit contains four protein chains arranged as a tetramer.

PHENIX³² was used for refinement, and COOT³³ was used for model building. The starting model for refinement of the C2 structures was prepared from the coordinates of wild-type ALDH7A1 complexed with NAD⁺ (PDB 4ZUK) or AA (PDB 4ZUL) by removing ligands and solvent, truncating the mutated residue and the catalytic Cys to Ala, and deleting the mobile C-terminus (last 12 residues). Initial crystallographic phases for the E427D-NAD⁺ structure were obtained by molecular replacement as implemented in MOLREP³⁴ via CCP4i³⁵ using a monomer search model prepared from the coordinates of wild-type ALDH7A1 complexed with NAD⁺ (PDB 4ZUK). Polder omit maps aided in modelling ligands and the 427-loop.³⁶ All the structures were validated using the PDB validation server and MolProbity.³⁷ Refinement statistics are listed in Table S2.

Partial occupancy of ligands was addressed as follows. NAD⁺ in E427Q-NAD⁺ was modelled as a single conformation with occupancy of 1.0 in four of the eight chains in the asymmetric unit. In the other four chains, dual conformations were modelled such that the occupancies of the A and B conformations summed to 1.0; the individual conformations have refined occupancies of 0.47 to 0.53. Similarly, the NAD⁺ in E427G-NAD⁺ was modelled as a single conformation in five chains (occupancy = 1.0) and as dual conformations in three chains (occupancy = 0.41–0.59). The NAD⁺ in E427D-NAD⁺ was modelled as a single conformation in all chains (occupancy = 0.86–0.93). The occupancy of AA in E427DAA was fixed at 1.0.

5.4 |. Small-angle X-ray scattering

Shutterless SAXS data collection was performed at beamline 12.3.1 of the Advanced Light Source through the SIBYLS Mail-in High Throughput SAXS program.³⁸ Prior to SAXS analysis, purified protein samples were passed over a Superdex 200 10–30 size-exclusion chromatography column in the presence of a buffer containing 50 mM HEPES (pH 8.0), 100 mM NaCl, 2% (v/v) glycerol, and 1 mM dithiothreitol. Samples were then supplemented with 10 mM NAD⁺ and dialysed overnight at 4°C against a buffer containing 50 mM HEPES (pH 8.0), 100 mM NaCl, 2% (v/v) glycerol, 1 mM dithiothreitol, and 10 mM NAD⁺.

SAXS data were collected on a Pilatus detector operating in shutterless mode, writing frames every 0.3 seconds. Buffer subtracted SAXS curves were averaged using SAXS FrameSlice by averaging the first 5 frames for the Guinier region (total 1.5 seconds), the first 10 second frames for the Porod region (total 3 seconds), and the first 20 frames for the high q region (total 6 seconds). PRIMUS¹⁹ was used to inspect the merged data and to derive SAXS parameters. The maximum particle dimension was estimated from calculations of the pair distribution function using GNOM³⁹ via PRIMUS. Theoretical SAXS curves were calculated using FoXS⁴⁰ and MultiFoXS.⁴⁰

Abbreviations:

AA

α-aminoadipate

AASAL

α-aminoadipate semialdehyde

ALDH

aldehyde dehydrogenase

ALDH7A1

aldehyde dehydrogenase 7A1

NMN

nicotinamide mononucleotide

P6C

Δ¹-piperideine-6-carboxylic acid

PA

L-pipecolic acid

PDE

pyridoxine-dependent epilepsy

PLP

pyridoxal 5’-phosphate