Phytoene Desaturase: Genes, Enzymes and Phylogenetic Aspects

J Plant Physiol. VoL

143. pp. 444-447 (1994)

Review

Phytoene Desaturase: Genes, Enzymes and Phylogenetic Aspects GERHARD SANDMANN Botanisches Institut, J. W. Goethe Universitat, P.O. Box 111932, D-60054 Frankfurt/M., Germany Received September 7,1993 . Accepted November 25,1993

Summary

Recent work on phytoene desaturases and also on r-carotene desaturase is presented and discussed. This involves functional complementation of phytoene desaturation by heterologous expression of different genes coding for this enzyme originating from Rhodobacter capsulatus, Erwinia uredovora, and Synechococcus and the enzymatic characterization of purified enzymes. The results obtained demonstrate that different phytoene desaturases exist with respect to the catalytic reaction and the protein sequence. The gene for the proposed r-carotene desaturase was cloned from the cyanobacterium Anabaena and the amino acid sequence deduced from the gene sequence compared to the other carotene desaturases. The phylogenetic relationship between different types of phytoene desaturases and the r -carotene desaturase is discussed. Introduction

A biosynthetic pathway leading to the formation of carotenoids can be found in heterotrophic bacteria and fungi where some species possess this biosynthetic capacity or among photosynthetic pro- and eukaryotes. In the latter lower and higher plants carotenogenesis is obligatory for their photosynthetic growth. The most universal carotenoid biosynthetic pathway is the sequence leading to the formation of (j-carotene which can be found in all the groups mentioned above (Sandmann, 1991). Conversion of phytoene, the first carotene of the pathway to (j-carotene, involves four desaturation steps leading to lycopene as the maximally desaturated carotene (Fig. 1). Since a few years, genes encoding phytoene desaturases are available from bacteria (Armstrong et al., 1989; Misawa et al., 1990), the fungus Neurospora (Schmidhauser et al., 1990), cyanobacteria (Chamovitz et al., 1991; Martlnez-Ferez and Vioque, 1992), and higher plants (Bartleyet al., 1991; Pecker et al., 1992; Hugueney et al., 1992). These genes were employed for the characterization of the enzymatic reactions carried out by the resulting polypeptides and the expression and purification of these enzymes.

Functional complementation For characterization of the products of the phytoene desaturase genes from Erwinia, Rhodobacter and Synechococcus, © 1994 by Gustav Fischer Verlag, Stuttgart

Phytoene

Phytofluene

~- Carotene

Neurosporene

lycopene

Fig. 1: Carotene desaturation sequence from phytoene to lycopene.

functional complementation in E. coli which lacks the potential to synthesize carotenoids and analysis of the produced carotenes were carried out. For this purpose, E. coli was cotransformed with two plasmids. One carried the genes crtE and crtE which are necessary for the synthesis of the substrate phytoene (Sandmann and Misawa, 1992) and a second plasmid carrying one of the phytoene desaturase genes. The resulting E. coli transformants were pigmented and HPLC analysis of accumulated carotenes was performed

Phytoene desaturases: genes and enzymes Table 1: Properties of phytoene desaturases from different organIsms. No. of desaturation steps

Cofactor requirement

Inhibitors

Synechococcus Phyt. I-Carotene

2

NAD(P)

Herbicides'

Rhodobacter Phyt. -

3

FAD

DPAb

4 (6)

FAD

DPAb

Source

Catalytic reaction

Phyt. -

Lycopene

, = bleaching herbicides like norflurazon, fluridone etc.

b = diphenylamine.

(Linden et al., 1991). It was shown that the reaction product of the Synechococcus desaturase is r-carotene. Three different isomers of this carotene with two double bonds additional to phytoene were detected of which all-trans r-carotene was the minor one. In contrast, the Rhodobacter enzyme introduced three double bonds yielding mainly all-trans neurosporene together with two cis isomers. In case of the Erwinia enzyme all-trans lycopene with four newly introduced double bonds was the major product but also a cis lycopene isomer and small amouts of bisdehydrolycopene (six additional double bonds) were detected. Very similar results were obtained in cell-free reactions in which phytoene was converted by membranes isolated from E. coli with phytoene desaturase genes from the three organisms (Sandmann et al., 1993). Other differences found between both bacterial and the cyanobacterial phytoene desaturase are their cofactor requirement and their inhibition properties. The Synechococcus enzyme uses NAD or NADP in the in vitro reaction which is inhibited by bleaching herbicides like norflurazon. In contrast, the reaction of the other bacterial phytoene desaturases is dependent on FAD and is inhibited by diphenyl amine (DPA). Obviously, there is a functional diversity of these three different types of phytoene desaturases which is reflected by

11

the number of desaturation steps carried out. Their properties are summarized in Table 1. Complementation of the phytoene desaturase gene from tomato resulted in accumulation of -carotene in E. coli as observed in the case of the cyanobacterium (Pecker et al., 1992) whereas the phytoene desaturase from the fungus Neurospora synthesized lycopene after complementation of its gene in Rhodobacter (Bartley et aI., 1990) resembling the Erwinia enzyme. Homology plots of the deduced amino acid sequence indicate the relationship of the proteins (Fig. 2). Comparing the amino acid sequence deduced from the tomato pds gene with the sequence from a green alga or a cyanobacterium a remarkably high homology is observed as indicated by the dotted lines. In contrast, homology to bacterial and fungal phytoene desaturases is negligible. The only region sharing homology with the pds type enzymes is near the N terminus and shows all the characteristics of a dinucleotide binding site (Pecker et aI., 1992). Nevertheless, the fungal and bacterial phytoene desaturases are highly conserved forming a second related group. The Rhodobacter enzyme although different to the Erwinia and Neurospora enzyme with respect to its reaction product is homologous to them. All the results demonstrate that during the course of evolution two completely different and unrelated phytoene desaturase proteins were acquired.

r

Neurosporene

Erwinza

445

Purification ofphytoene desaturases Purification of phytoene desaturase from plant tissue is very difficult to achieve. Nevertheless, this enzyme was isolated from Capsicum chromoplasts and purified (Hugueney et aI., 1992). However, when appropriate genes are available, it is much easier to make an overexpressing gene construct and express large quantities in E. coli. This approach was successful for several carotenogenic enzymes including phytoene desaturases from Erwinia and Synechococcus. For expression of phytoene desaturase a plasmid was constructed

DUNALIELLA

SYNECBOCOCCUS 585 1 474 1

ERWINIA

NEUROSPORA

492 1

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595

.

o

:;;

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o

, \

583

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\,,1

r-------~------~------~------~

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Fig. 2: Homology plots of deduced amino acid sequences of phytoene desaturases from different organisms (Peeker et aI., 1992).

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446

GERHARD SANDMANN

by cloning the entire coding region of the crt! gene from Erwinia behind the lacZ promoter of pUe18 resulting in a reading frame for the full polypeptide with additional 9 amino acids at the N terminus (Fraser et al., 1992). In E. coli transformants this plasmid mediated the expression of phytoene desaturase to a final concentration of about 10 % of total cellular protein. Under these conditions, the recombinant protein is sequestered in inclusion bodies where it can be solubilized from by urea treatment. Subsequent purification to homogeneity involved OEAE-cellulose chromatography and 50S-polyacrylamide gel electrophoresis. Due to the high expression rate it is very easy to obtain several mg of the protein by this purification procedure. As a consequence of the urea treatment, the resulting enzyme is poorly active. However, it was possible to regain its activity by removal of the denaturant and dilution of the sample in the presence of OTT which allows refolding of the enzyme. The Erwinia phytoene desaturase has a molecular weight of 56.2 kd and catalyzed as expected the conversion of 15-cis phytoene to trans lycopene and also to some extent to bisdehydrolycopene. FAD was involved as cofactor in the desaturation reaction of this bacterial type of phytoene desaturase. The same strategy was followed in the purification of the phytoene desaturase from Synechococcus (Fraser et al., 1993) and several mg of the homogenous enzyme were also obtained. This 53 kd membrane protein could be reactivated after lipid replenishment. Inhibition was observed by several bleaching herbicides (Sandmann and Fraser, 1993). The co-

Neurospora (ai-I) ,

,

..

,

Fig. 3: Alternative ways for the desaturation sequence from phytoene to lycopene.

factors for this cyanobacteriall algal/plant type phytoene desaturase were either NAD or NADP whilst FAD was ineffective as electron acceptor. The dependence of both purified phytoene desaturases on oxidized dinucleotides as electron and hydrogen mediators confirms a dehydrogenase-electron transferase mechanism for the desaturation reaction as proposed by Goodwin (1983).

Cloning of r-carotene desaturase and phylogeny of Carotene Desaturases As indicated in Fig. 3, there are two alternatives in the desaturation pathway from phytoene to lycopene. One way is catalyzed by a single enzyme, the crt! product, and the other proceeds via r-carotene in a two step reaction involving a second desaturase. The gene zds encoding r -carotene desatur-

.

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.. .

,

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,

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Rhodobacter (ertl)

Erwinia (ertl) .. ... ...

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Anabaena (zds) ,

~dS

...

Rhodobaeter (ertD)

...

fl-Carotene

~-Carotene

, ~

Lycopene -

PdS~

..

'.

,.,

+

- - Phytoene

.•

,

,

Anabaena (zds)

,

I

crt I

.:.

., ."

Syneehococcus (pds)

Glycine (ptb)

...

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Fig. 4: Protein homology plots of r-carotene desaturase (zds) in comparison to various carotenoid desaturases.

!P

...

Phytoene desaturases: genes and enzymes

ase was cloned recently by heterologous complementation in E. coli in a similar way as described above. Cells of E. coli were cotransformed with two plasmids, one carrying all genes necessary for the synthesis of the substrate t-carotene, the other was from an Anabaena library. The positive clone was selected by its color change from yellow to red due to lycopene synthesis catalyzed by the product of the new gene (Linden et aI., 1993). The sequence was obtained recently (Linden et aI., submitted for publication). Figure 4 presents the amino acid comparison of r -carotene desaturase with carotenoid desaturases from different species using protein homology plots. The best homology was found with the crt! phytoene desaturase genes from bacteria and fungi and a methoxyneurosporene desaturase gene crtD (Armstrong et aI., 1989) from Rhodobacter. The highest homology was 29 % identity for the Neurospora phytoene desaturase. In contrast, the homology between the r-carotene desaturase gene and the pds phytoene desaturase genes from cyanobacteria and higher plants was comparable weak. It can be seen from the homology plots (Fig. 4) that the amino acid homology of r-carotene desaturase to the latter is mainly limited to the N terminus of the polypeptides. As indicated by quantitative homology calculations, the following ranking of the structural conservation of r-carotene desaturase to other desaturases could be established: al·J, Neuros-

pora > crt!, Erwinia > crtD, Rhodobacter > crt!, Rhodobac· ter » pds, Glycine > pds, Synechococcus.

All available data indicate that the two protein types of phytoene desaturases do not have a common ancestor and that they were acquired through convergent evolution (Pecker et aI., 1992). A consequence of the replacement of the four-step desaturase crt! by a two-step desaturase pds in the course of evolution is the necessity for the presence of a r-carotene desaturase in all pds containing organisms (Linden et al., 1993), as indicated in Figure 2. Based on the homology plots in Figure 4, it can be proposed that the newly appearing r-carotene desaturase gene was phylogenetically derived from the crt! phytoene desaturase gene of bacteria and fungi. This specialization occurred parallel to the evolution of oxygenic photosynthesis. Acknowledgement

This work was supported by the German-Israeli Foundation for Scientific Research and Development (GIF) and a grant from the EC Biotechnology Programme.

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447

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