Cloning, functional expression and characterization of a bifunctional 3-hydroxybutanal dehydrogenase /reductase involved in acetone metabolism by Desulfococcus biacutus

Table 1 Specific NAD(H)-dependent oxidoreductase activities determined for the heterologously expressed and purified Debia-MDR protein

Reduction with NADH			Oxidation with NAD⁺
Substrate¹⁾		Spec. activity mU mg⁻¹	Substrate²⁾		Spec. activity mU mg⁻¹
	Formaldehyde	b.d.		Methanol	n.d.
	Acetaldehyde	52 ± 14		Ethanol	73 ± 13
	Propanal	380 ± 15		1-Propanol^b	22 ± 2
	Butanal	301 ± 24		1-Butanol	47 ± 15
	Isobutanal	276 ± 30		Isobutanol	n.d.
	Pentanal	325 ± 35		1-Pentanol	11 ± 3
	Benzaldehyde	b.d.		Benzyl alcohol	n.d.
	Propanone (Acetone)	93 ± 2		2-Propanol^a (Isopropanol)	21 ± 1
	Butanone	65 ± 11		2-Butanol^b	115 ± 8
	2-Pentanone	126 ± 38		2-Pentanol	n.d.
	3-Pentanone	141 ± 19		3-Pentanol	n.d.
	2-Hexanone	45 ± 9		2-Hexanol	n.d.
	3-Hydroxybutanone (Acetoine)	326 ± 38		2,3-Butanediol	150 ± 8
	2,3-Butandione (Diacetyl)	298 ± 42		3-Hydroxybutanone (Acetoine)	b.d.
	3-Hydroxybutanal	248 ± 59		1,3-Butanediol	80 ± 23
	4-Hydroxy-2-butanone	155 ± 31		1,3-Butanediol	80 ± 23
	3-Oxobutanal (Acetoacetaldehyde)	n.s.		3-Hydroxybutanal	83 ± 18
	3-Oxobutanal (Acetoacetaldehyde)	n.s.		4-Hydroxy-2-butanone	18 ± 3

b.d. below detection limit (<1 mU mg⁻¹ protein), n.d. not determined, n.s. no substrate was available for testing
¹⁾Assay conditions: anoxic 25 mM MOPS buffer (pH 7.2) plus 3 mM DTT and 50 μM ZnCl₂, 30 °C. Reactions in reductive direction were assayed with 0.1 mM NADH. Reactions were started by addition of 5 mM substrate
²⁾Assay conditions: anoxic 25 mM MOPS buffer (pH 8.0) plus 3 mM DTT and 50 μM ZnCl₂, 30 °C. Reactions in the oxidative direction were assayed with 2.5 mM NAD⁺, or at pH 7.2 with 0.5 mM NAD⁺(^a), or at pH 7.2 with 2.5 mM NAD⁺(^b)

ISSN: 1471-2180