Characterisation of recombinant human fatty aldehyde dehydrogenase: Implications for Sjögren-Larsson syndrome

Figures & data

Figure 1 a) Model of the secondary structure of FALDH highlighting the presence of the Rossmann fold; b) The hydrophobic pocket of FALDH generated from the molecular graphics package, YASARA 35-37. The substrate has been proposed to insert into the cleft to aid catalysis by association with the active site residue, Cys-241 (underlined, centre).

Table I.  1: FALDH (4 μg) activity with straight-chain substrates (50 μM), in the presence of N-lauroylsarcosine (C5-C12) or Triton X-100 (C14–C18).

	Condition	Rate (nmol/min/mg protein ± S.E.M.)
1	Pentanal (C5:0)	43 ± 19
2	Heptanal (C7:0)	227 ± 7
3	Octanal (C8:0)	133 ± 3
4	Decanal (C10:0)	150 ± 17
5	Dodecanal (C12:0)	163 ± 3
6	Tetradecanal (C14:0)	183 ± 19
7	Hexadecanal (C16:0)	195 ± 25
8	Octadecanal (C18:0)	180 ± 25

Table II.  2: Kinetic parameters (K_m, V_max and k_cat) for various straight- and branched-chain aldehyde substrates using 0.5 μg FALDH.

	Substrate	Km (μM)	Vmax (nmol/min/mg protein)	kcat (s^− 1)	kcat/Km (M^− 1 s^− 1)
1	Decanal	3.8	2287	2.18	5.73 × 10^5
2	Dodecanal	13.6	2335	2.23	1.64 × 10^5
3	Tetradecanal	10.3	900	0.86	0.58 × 10^5
4	Hexadecanal	8.3	988	0.95	1.14 × 10^5
5	Octadecanal	20.0	1594	1.52	0.76 × 10^5
6	Farnesal	23.0	972	0.93	0.40 × 10^5
7	cis-11-Hexadecenal	34.3	1466	1.40	0.40 × 10^5
8	NAD^+	180	1338	1.28	0.07 × 10^5

Figure 2 The three stages of the proposed mechanism for aldehyde oxidation: 1. attack of the Cys-241 thiolate anion on the aldehyde carbonyl. 2. Rearrangement of the thiohemiacetal intermediate and hydride transfer to NAD⁺. 3. Hydrolysis of the thioester to regenerate Cys241 and generate the respective acid. R = fatty acid side-chain. ADPr, Adenosine diphosphate ribose;

Figure 3 The proposed concerted mechanism of alcohol oxidation by FALDH. Removal of the OH proton by the general base imidazole (of His) leads to transfer of a hydride onto the nicotinamide ring and formation of the aldehyde.