Part:BBa_K1324001

W176Q E. coli Aldehyde Dehydrogenase

This part encodes the aldehyde dehydrogenase enzyme from Escherichia coli with the W176Q mutation. This enzyme catalyzes the reaction of an aldehyde and NAD+ into the corresponding carboxylic acid and NADH.

Kinetic Constants of W176V Escherichia coli Aldehyde Dehydrogenase
Substrate	kcat (s-1)	kcat Error (s-1)	KM (uM)	KM Error (uM)	Ki (uM)	Ki Error (uM)	kcat/KM (M-1s-1)	kcat/KM Error (M-1s-1)
Propionaldehyde	7.57	1.24	2575.71	559.82	N/A	N/A	2937.45	800.31
Butyraldehyde	4.42	0.12	532.70	32.00	N/A	N/A	8306.37	546.34
Pentanal	5.44	0.35	165.35	35.25	N/A	N/A	32877.93	7328.72
Hexanal	7.29	0.37	38.93	8.48	N/A	N/A	187159.14	41828.31
Heptanal	12.46	1.76	100.89	23.73	591.10	152.70	123500.84	33883.89
Octanal	16.21	5.97	301.64	146.69	547.52	335.18	53752.82	32794.34
Nonanal	10.45	1.88	161.83	45.71	1292.60	584.89	64574.73	21618.61
Decanal	6.88	0.71	91.54	17.33	1764.75	570.99	75154.88	16187.92
E-2-Pentenal	ND	ND	ND	ND	ND	ND	ND	ND
E-2-Hexenal	0.22	0.02	28.11	9.85	N/A	N/A	7797.24	2796.55
E-2-Octenal	N/A	N/A	N/A	N/A	N/A	N/A	2056.70	237.60
E-2-Nonenal	N/A	N/A	N/A	N/A	N/A	N/A	1554.50	261.50
E-2-Decenal	N/A	N/A	N/A	N/A	N/A	N/A	1299.10	210.50
E,E-2,4-Heptadienal	ND	ND	ND	ND	ND	ND	ND	ND
E,E-2,4-Nonadienal	ND	ND	ND	ND	ND	ND	ND	ND
E,E-2,4-Decadienal	ND	ND	ND	ND	ND	ND	ND	ND

Key:
N/A: Not applicable
ND: No detection of activity

Stock Solutions (Unless stated, all solutions were prepared in 11.1mM Potassium Phosphate, 111.1mM Potassium Chloride solution)
Base: 1.54% Tween 20 (v/v), 1.11mM Dithiothreitol (DTT)
Substrate: 10x final concentration of aldehyde in isopropyl alcohol
Enzyme: 0.004mg/mL Aldehyde Dehydrogenase enzyme, 4mM NAD+, 1.11mM Dithiothreitol (DTT)

Order of addition to initiate assay: (1) 130uL substrate, (2) 20uL aldehyde, (3) 50uL enzyme. Assays were conducted in a Costar 96 well flat bottom plate. The activities of all aldehyde dehydrogenases were measured by the accumulation of NADH resulting from the oxidation of the assayed substrates. All assays were performed by incubating 18.72nM aldehyde dehydrogenase enzyme with substrate in a 200µL reaction containing 10mM phosphate buffer containing 100mM potassium chloride (pH 7.4), 1mM NAD+, 1mM DTT, 1% Tween 20, 10% isoproyl alcohol, and substrate. All aldehyde dehydrogenases were assayed against substrate concentrations of 1000uM, 500uM, 100uM, 50uM, 10uM, and 5uM. The change in absorbance of NADH at a wavelength of 340nm was measured using Biotek Epoch and Synergy microplate spectrophotometers. Kinetic constants were derived from the steady-state velocity curves using R. Curves were fit linearly [V=(kcat/KM)S], Michaelis-Menten [V=Vmax*S/(Km+S)], or substrate inhibition [V=Vmax*S/(Km+S*(1+S/Ki))]. Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

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• 23
  COMPATIBLE WITH RFC[23]
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  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]