Condiciones de los factores productivos

The recent solving of the crystal structures of a class 1 AlDH (S. Moore et ai,

manuscript in progress), and a class 2 AlDH (Steinmetz et al., 1 997), can b e used in addition with the above multiple sequence alignment, to explain the differing substrate specificities of these two classes of enzymes. In particular, the reasons for the inability of AlDH 2 enzymes to accept retinal as a substrate, while the AlDH 1 enzymes can oxidise retinal may be addressed . The tertiary structures o f the two enzymes - in particular the substrate binding pockets - are very similar. However, there are two amino acid substitutions between the classes which may explain the differences in substrate specificities. Residues which form the binding pocket (sheep AlDH 1 numbering) are: Met 1 20, Phe 1 70, Leu 1 73 , Met 1 74, Trp 1 77, Tyr 296, ne 3 03, Val 459, and Phe 465. All of these residues except two (ne 3 03 and Va1 459) are highly conserved between class 1 and class 2 enzymes. However, position 459 in the class 1 enzymes (except E. coii, yeast and chicken enzymes) is always a small hydrophobic residue, e.g. valine in the sheep, while in the class 2 enzymes the equivalent amino acid is a phenylalanine. Position 303 in the class 1 enzymes is always a small hydrophobic residue, while in the class 2 enzymes it is generally a cysteine (except the horse class 2 (H02), where it is Gly) . The introduction of a larger side chain (Phe) and/or a difference in charge in the binding pocket (with the substitution of a Cys for a small hydrophobic residue) may change the ability of the enzyme to make interactions with substrates and hence, may alter the ability of apparently similar enzymes to bind the large hydrophobic substrate retinal.

5.4 Conclusions

The multiple sequence alignment clarifies relationships between a large number of aldehyde dehydrogenases, of particular interest those with the ability to oxidise retinal. The four retinal-specific aldehyde dehydrogenases (RALDHs) recently isolated from rat and mouse tissues (Bhat et al. , 1 995; Penzes et al. , 1 997; Wang et al. , 1 996; Zhao et al., 1 996) have been proposed to play a maj or role in retinal metabolism. However, due to the lack heretofore of comparison between these and other aldehyde dehydrogenases

5 - 1 27

with the ability to oxidise retinal, it has been difficult to determine the relative role played by each protein in retinoic acid production. The sequence alignment has shown that the four cloned RALDH genes (RalDHl , RALDH- l , RalDH2 and RALDH-2), in all likelihood encode only two distinct proteins. The type 1 RALDHs show high similarity with class 1 aldehyde dehydrogenases, some of which have been demonstrated to oxidise retinal ( S I , H I , M l ), and would be classified as class 1 enzymes on this basis. The type 2 RALDHs clustered most closely with the class 1 enzymes; however, they show significant sequence differences and may not be true class 1 enzymes. It has also been shown that type 2 RALDH enzymes do not oxidise some aldehyde substrates such as acetaldehyde, propanaldehyde and benzaldehyde (Wang et al. , 1 99 6), and hence may be more retinal-specific enzymes than S I , H I , MI and RALDH type I enzymes, and may have a different role.

In addition, the basis for the differing substrate specificities of the class 1 and 2 enzymes with respect to retinal has been proposed, based on the conservation of residues found in the substrate binding pocket. The elucidation of the crystal structure of sheep AlDH 1 ( S . Moore, Massey University), and the recent publication of the structure of bovine AlDH 2 (Steinmetz et al. , 1 997), identified residues important in substrate binding and forming the binding pocket (see section 5 . 3 . 1 ) . On the basis of a comparison of the class 1 and 2 structures and multiple sequence alignment, we have proposed that two residues located in the substrate binding pocket are responsible for the ability of only class 1 aldehyde dehydrogenases to bind and oxidise retinal; IIe 3 03 and Val 459 (usually Cys 303 and Phe 4 5 9 in the class 2 enzymes). Further crystallographic modelling studies currently being carried out may clarify this important difference in substrate specificity ( S . Moore, manuscript in progress). With the large number of isolated and sequenced mammalian AlDHs, an alignment such as this one is invaluable when looking at sequence and structural motifs, conserved residues, substrate specificities, relationships between enzymes, and in the classification of new AlDHs.

6.

Chapter Six:

In Vivo Studies o n Retinal

Dehydrogenation

6.1 Introduction and Aims

A human neuroblastoma cell line, SH-SYSY (Biedler et aI., 1 973), was used to attempt to study the in vivo production of retinoic acid from retinal. This cell line was chosen because it had previously been demonstrated to be responsive to retinoic acid (Pahlman

et aI. , 1 984) and because of its availability. The SH-SYSY cell line is a neuroblast- or N-type cell line derived from SK-N-SH cells. Culturing SH-SYSY cells in the presence of all-trans retinoic acid induces morphological conversion of the cells into a neuronal­ like phenotype, with the extension of cell processes known as neurites. B ecause of the responsiveness of this cell line to retinoic acid, it was thought likely that enzymes involved in retinoic acid synthesis would be present. The main aim of this work was to develop a system using SH-SYSY cells, whereby the effects o f ethanol and acetaldehyde on the conversion of retinal to retinoic acid could be examined. This work could have potential importance with regards to the mechanisms by which fetal alcohol syndrome arises (see section 1 . 5).

6.2 Results