Dihydroxyacetone Reductase from Mucor javanicus. 1. Isolation and Properties

Abstract

An NADPH-dependent oxidoreductase has been extracted from the mycelium of the fungus Mucorjavanicus (Wehmer) and enriched 1000-fold with respect to the protein contained in the crude extract after centrifugation at 260Uxg. The molecular weight of the enzyme was estimated by gel filtration to be about 100000; electrophoresis under dissociating conditions indicates four subunits of molecular weight about 28000. Data on stability and activity of the enzyme as a function of pH and temperature are reported. From a kinetic study and product analysis of the reduction of the two enantiomeric trans-I-decalones and also from a kinetic study of the oxidation of the two diastereomeric pairs of trans-1-decalols it follows that the enzyme is an e-Si oxidoreductase (according to the nomenclature proposed by Dutler et id., Eur. J. Bioclzem. 22 [1971] 203 -21 2 and Prelog and Helmchen, Helv. Clzim. Acta, 55 [1972] 2581 -2598). This classification is amply confirmed by the kinetic behaviour of a large number of alicyclic substrates. Using (4-2Hsi)-labelled coenzyme to reduce (9s)-trans-l,4-decalindione, it was shown that the enzyme is Hsi ( = Hs = HH)-stereospecific with respect to the coenzyme. It is demonstrated that the oxidoreductase from Mucor javunicus can be used for the preparation of optically pure chiral alcohols and ketones. In the following paper evidence is presented that the natural substrate of the enzyme is dihydroxyacetone.In the course of work carried out in our laboratory on the stereochemistry of enzyme-catalyzed oxidoreductions [ 1 -41 a new NADPH-dependent oxidoreductase was isolated from the fungus Mucor ,jawnicus (Wehmer). The enzyme was active towards several aliphatic and alicyclic ketones and the corresponding alcohols. The isolation of dihydroxyacetone from the mycelium extract and the finding that it is a highly active substrate, strongly suggested that the enzyme is a dihydroxyacetone reductase. Evidence in favour of this assumption is presented in the following paper [5].Preliminary experiments with a few alicyclic ketones had shown that the new enzyme transfers hydrogen exclusively to the Si face of the carbonyl. The steric course of hydrogen transfer is thus opposite to that previously reported for reductions catalyzed by other similar oxidoreductases, all of which transfer hydrogen to the Re face [4-71. It was therefore of interest to establish the steric course of the reactions catalyzed by the Mucor enzyme for rigid substrates such as trans-I -decalones and trans-I-decalols. Kinetic ~-Ahh,o~ia/ion. NMR, nuclear magnetic resonance. E 1 1~~7 7 1~.

Purification and Properties of NADP+-linked Glycerol Dehydrogenase from Neurospora crassa

“…First, a relatively substrate-specific type which reversibly catalyses the conversion of glycerol to glyceraldehyde (Toews, 1966(Toews, ,1967Sheys et al, 1971 ;Kormann et al, 1972) or to dihydroxyacetone (Ben-Amotz & Avron, 1973;Dutler et al, 1977;Hochuli et al, 1977), and secondly, a non-specific type called aldehyde reductase which not only catalyses the reduction of aldoses such as glyceraldehyde but also oxidizes several sugar alcohols (polyols) such as D-sorbitol, L-arabitol and glycerol (Bosron & Prairie, 1972). We have detected high levels of NADP+-linked polyol dehydrogenase activity with sorbitol, mannitol, xylitol and glycerol in crude extracts of Neurospora crassa.…”

Purification and Properties of Two Different Dihydroxyacetone Reductases inGluconobacter suboxydansGrown on Glycerol

“…Judging from the Km values for DHA, NADPH-DHAR may have stronger affinity to DHA than does NADH-DHAR. Differently from the two DHARs in G. suboxydans IFO 3255 studied here, NADP-dependent glycerol dehydrogenase (EC 1.1.1.156) from Dunaliella parva 10,11) and Mucor javanicus 13) are reversible in glycerol oxidation and DHA reduction at almost equal reaction rates. Their pH optima for DHA reduction and for glycerol oxidation are observed at pH 6-7 and 9-10.…”

“…Five different glycerol oxidizing enzymes are known: glycerol: NAD 2-oxidoreductase DHA-forming (EC 1.1.1.6), [1][2][3][4][5][6] glycerol: NADP 1-oxidoreductase glyceraldehyde (GAL)-forming (EC 1.1.1.72), [7][8][9] glycerol: NADP 2-oxidoreductase DHA-forming (EC 1.1.1.156), [10][11][12][13] glycerol: acceptor 2-oxidoreductase DHA-forming (EC 1.1.99.22), [14][15][16][17][18] and glycerol: oxygen 1-oxidoreductase GAL-forming (EC 1.1.3.99). [19][20][21] These enzymes are divided into two groups: GALforming and DHA-forming.…”

“…[19][20][21] These enzymes are divided into two groups: GALforming and DHA-forming. With respect to DHAforming glycerol dehydrogenase (GLDH), the NADdependent enzyme has been extensively studied, while only a few preliminary papers are available on the NADP-dependent enzyme, and only from eukaryotic microorganisms such as halophilic alga Dunaliella parva [10][11][12] and Mucor javanicus, 13) not from prokaryotes. Differently from NAD(P)-dependent DHA-forming dehydrogenases, PQQ requiring GLDH is membrane-bound and is localized on the outer surface of the cytoplasmic membrane of acetic acid bacteria, functioning in the periplasmic space, to which industrial DHA production is formulated.…”

“…As itemized above, several reports on NADP-dependent GLDH (EC 1.1.1.156) are available for eukaryotes such as the halophilic green alga Dunaliella parva [10][11][12] and Mucor javanicus, 13) although they have been only partially purified. It is important to search NADPHdependent DHAR (synonymous with NADP-dependent GLDH) and NADH-dependent DHAR (synonymous with NAD-dependent GLDH) in acetic acid bacteria, because there is no clear evidence as to which enzyme is responsible for allowing Gluconobacter strains to grow on glycerol medium.…”