Tài liệu Báo cáo khoa học: Enzymes for the NADPH-dependent reduction of dihydroxyacetone and

Enzymes for the NADPH-dependent reduction of

dihydroxyacetone and D-glyceraldehyde and

L-glyceraldehyde in the mould Hypocrea jecorina

Janis Liepins1,2, Satu Kuorelahti1

, Merja Penttila¨

1 and Peter Richard1

1 VTT Biotechnology, Espoo, Finland

2 University of Latvia, Institute of Microbiology and Biotechnology, Riga, Latvia

Dihydroxyacetone (DHA), d-glyceraldehyde and

l-glyceraldehyde can be reduced using NADPH as a

cofactor to form glycerol and NADP. Enzymes cataly￾sing this reaction are generally called NADP:glycerol

dehydrogenases. NADP:glycerol dehydrogenase activ￾ity is common in moulds and filamentous fungi.

Enzymes from different species of filamentous fungi

have been purified and characterized. The enzymes

purified from Aspergillus niger [1] and Aspergillus nidu￾lans [2] catalyse the reversible reaction from glycerol

and NADP to DHA and NADPH. For the A. niger

enzyme, an equilibrium constant of 3.1–4.6 · 10)12 m

was estimated for the reaction:

Glycerol þ NADP Ð DHA þ NADPH þ Hþ

A glycerol dehydrogenase with slightly different prop￾erties was described in Neurospora crassa, where

d-glyceraldehyde was the preferred substrate over

DHA in the reductive reaction. This enzyme was also

reversible, i.e. it showed activity with glycerol and

NADP [3]. The purified glycerol dehydrogenases from

A. nidulans and A. niger also showed low activity with

d-glyceraldehyde; however, DHA was the preferred

substrate [2]. The A. niger enzyme was commercially

available as a partly purified preparation, and partial

amino acid sequences were available [4].

Keywords

dihydroxyacetone; glycerol dehydrogenase;

Hypocrea jecorina; L-glyceraldehyde; NADP￾specific glycerol dehydrogenase

Correspondence

P. Richard, VTT, Tietotie 2, Espoo,

PO Box 1000, 02044 VTT, Finland

Fax: +358 20 722 7071

Tel: +358 20 722 7190

E-mail: [email protected]

(Received 4 May 2006, revised 7 July 2006,

accepted 17 July 2006)

doi:10.1111/j.1742-4658.2006.05423.x

The mould Hypocrea jecorina (Trichoderma reesei) has two genes coding

for enzymes with high similarity to the NADP-dependent glycerol dehy￾drogenase. These genes, called gld1 and gld2, were cloned and expressed in

a heterologous host. The encoded proteins were purified and their kinetic

properties characterized. GLD1 catalyses the conversion of d-glyceralde￾hyde and l-glyceraldehyde to glycerol, whereas GLD2 catalyses the con￾version of dihydroxyacetone to glycerol. Both enzymes are specific for

NADPH as a cofactor. The properties of GLD2 are similar to those of the

previously described NADP-dependent glycerol-2-dehydrogenases

(EC 1.1.1.156) purified from different mould species. It is a reversible

enzyme active with dihydroxyacetone or glycerol as substrates. GLD1

resembles EC 1.1.1.72. It is also specific for NADPH as a cofactor but has

otherwise completely different properties. GLD1 reduces d-glyceraldehyde

and l-glyceraldehyde with similar affinities for the two substrates and sim￾ilar maximal rates. The activity in the oxidizing reaction with glycerol as

substrate was under our detection limit. Although the role of GLD2 is to

facilitate glycerol formation under osmotic stress conditions, we hypothes￾ize that GLD1 is active in pathways for sugar acid catabolism such as

d-galacturonate catabolism.

Abbreviations

DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate.

FEBS Journal 273 (2006) 4229–4235 ª 2006 The Authors Journal compilation ª 2006 FEBS 4229