Plant Molecular Biology 54: 39–54, 2004. 39 © 2004 Kluwer Academic Publishers. Printed in the Netherlands. A small family of LLS1-related non-heme oxygenases in plants with an origin amongst oxygenic photosynthesizers John Gray1,∗, Ellen Wardzala1,ManliYang1, Steffen Reinbothe2, Steve Haller1 and Florencia Pauli3 1Department of Biological Sciences, University of Toledo, 2801 West Bancroft Street, Toledo, OH 43606, USA (∗author for correspondence; email [email protected]); 2Laboratoire de G´en´etique Mol´eculaire des Plantes, Universit´e Joseph Fourier et CNRS, CERMO, BP 53, 38041 Grenoble, France; 2Department of Genetics, School of Medicine, Stanford University, Stanford, CA 94305, USA Received 22 July 2003; accepted in revised form 22 December 2003 Key words: Acd1, Cao (chlorophyll a oxygenase), Cmo (choline monooxygenase), dioxygenase, Lls1, non-heme oxygenase, Pao (pheophorbide a oxygenase), Ptc52, Tic55 Abstract Conservation of Lethal-leaf spot 1 (Lls1) lesion mimic gene in land plants including moss is consistent with its recently reported function as pheophorbide a oxygenase (Pao) which catalyzes a key step in chlorophyll degrada- tion (Pruzinska et al., 2003). A bioinformatics survey of complete plant genomes reveals that LLS1(PAO) belongs to a small 5-member family of non-heme oxygenases defined by the presence of Rieske and mononuclear iron- binding domains. This gene family includes chlorophyll a oxygenase (Cao), choline monooxygenase (Cmo), the gene for a 55 kDa protein associated with protein transport through the inner chloroplast membrane (Tic55) and a novel 52 kDa protein isolated from chloroplasts (Ptc52). Analysis of gene structure reveals that these genes diverged prior to monocot/dicot divergence. Homologues of LLS1(PAO), CAO, TIC55 and PTC52 but not CMO are found in the genomes of several cyanobacteria. LLS1(PAO), PTC52, TIC55 and a set of related cyanobacterial homologues share an extended carboxyl terminus containing a novel F/Y/W-x2-H-x3-C-x2-C motif not present in CAO. These proteins appear to have evolved during the transition to oxygenic photosynthesis to play various roles in chlorophyll metabolism. In contrast, CMO homologues are found only in plants and are most closely related to aromatic ring-hydroxylating enzymes from soil-dwelling bacteria, suggesting a more recent evolution of this enzyme, possibly by horizontal gene transfer. Our phylogenetic analysis of 95 extant non-heme dioxygenases provides a useful framework for the classification of LLS1(PAO)-related non-heme oxygenases. Introduction the recent finding that the Arabidopsis thaliana Lls1 gene encodes pheophorbide a oxygenase (PAO) which The Lls1 gene was originally cloned from maize and catalyzes a key step in chlorophyll degradation (Fig- the absence of this gene function results in a light- ure 1D) (Pruzinska et al., in press). Since the discovery dependent cell death phenotype mediated by chloro- of LLS1 (PAO) in plants a few other genes have been plasts (Gray et al., 1997, 2002). We have found identified in plants that exhibit the same non-heme that this cell protective function is conserved between iron-binding motifs (Caliebe et al., 1997; Tanaka monocots and dicots (Yang et al., submitted). Based et al., 1998). In this study it is established that there are on the presence of two non-heme iron-binding mo- a total of five Lls1(Pao)-related genes in plants. The tifs conserved amongst aromatic ring-hydroxylating phylogenetic relationships between these Lls1(Pao)- enzymes in bacteria it was predicted that the Lls1 related genes and homologous bacterial enzymes were gene encodes an oxygenase function (Gray et al., examined in detail by comparing 95 known and pre- 1997, 2002). This prediction has been confirmed by dicted non-heme oxygenases. 40 Figure 1. Examples of known catalytic functions of non-heme oxygenases from bacteria and plants. A. Naphthalene dioxygenase from Pseudo- monas sp. strain G7 catalyzes the conversion of naphthalene to cis-1,2-dihydroxy-1,2-dihydronaphthalene. B. Choline monooxygenase (CMO) catalyzes the first step in the conversion of choline to the osmoprotectant glycine betaine in plant chloroplasts. C. Chlorophyll a oxygenase (CAO) catalyzes the first step in the conversion of the Chl a to Chl b. D. Pheophorbide a oxygenase (PAO) catalyzes the oxygenolytic opening of pheophorbide a at the α-mesoposition between C4 and C5 to produce a red chlorophyll catabolite (RCC). Non-heme iron oxygenases or hydroxylases that incorporation of both atoms of molecular oxygen are incorporate one or two atoms of dioxygen into sub- referred to as dioxygenases. These dioxygenases com- strates are found in many metabolic pathways (Lange prise a large and diverse group of multi-component en- and Que, 1998; Moraswki et al., 2000; Prescott zymes that play important roles in pathways as diverse and Lloyd, 2000; Ryle and Hausinger, 2002). En- as antibiotic synthesis to the degradation of aromatic zymes that incorporate only one atom of dioxygen compounds. The comparison of the deduced amino into substrates are termed monooxygenases (or mixed- acid sequences of numerous oxygenases is permitting function oxygenases). Oxygenases that catalyze the the evolutionary relationships between these enzymes 41 to be determined (Lange and Que, 1998; Prescott and classification scheme that groups these enzymes based Lloyd, 2000; Ryle and Hausinger, 2002). Of particular on the phylogenetic comparison of their terminal oxy- relevance to this study, because of their homology to genase components. This system, which is simple and the LLS1(PAO)-like plant oxygenases, are a group of powerful, is referred to here as the Nam classifica- microbial oxygenases that participate in the aerobic tion system. The Nam system classifies ARH enzymes degradation of aromatic hydrocarbons. These oxy- into four groups and representative examples included genases known as aromatic ring hydroxylases (ARHs) in this study are listed in Supplementary Table 1. In catalyze the hydroxylation of the aromatic ring as a this study we adapt and extend the Nam scheme for first step in the degradation of these compounds by classifying non-heme oxygenases to include plant and soil bacteria (Batie et al., 1991; Harayama et al., cyanobacterial non-heme oxygenases. 1992; Mason and Cammack, 1992; Jiang et al., The evolutionary relationship of plant LLS1-like 1996; Nam et al., 2001). An example is naph- oxygenases and cyanobacterial oxygenases to these thalene dioxygenase (NDO) from the soil bacterium microbial ARH enzymes is examined in this paper. Pseudomonas which oxidizes naphthalene to cis-1,2- Two of the plant oxygenases related to LLS1 have dihydroxy-1,2-dihydronaphthalene (Figure 1A). NDO previously defined biochemical functions and these has been crystallized permitting the structure and reac- are choline monooxygenase (CMO) and chlorophyll tion mechanism to be studied in detail (Kauppi et al., a oxygenase (CAO) (Figure 1B and C). Both oxy- 1998; Karlsson et al., 2003). NDO consists of two genases do not utilize phenolic compounds as sub- subunits (α and β) in a hexameric α3β3 composi- strates. CMO is a ferredoxin-dependent enzyme that tion. The α subunit contains a Rieske[2Fe-2S] center catalyzes the first step in two-step oxidation of choline and mononuclear iron at the active site. The Rieske to the osmoprotectant glycine betaine (Figure 1B). domain exhibits a conserved iron-binding domain, This enzyme is found as a homodimer in the chloro- C81-x-H83-x17-C101-x2-H104, which contrasts with plast stroma (Rathinasabapathi et al., 1997). CMO is the Rieske domain of chloroplast-type ferredoxins, in unique to plants: in bacteria (including halotolerant which four cysteine residues co-ordinate a [2Fe-2S] cyanobacteria) and mammals that synthesize glycine center. In the NDO Rieske [2Fe-2S] center the Fe1 betaine, this first step is catalyzed by choline dehydro- is coordinated by Cys-81 and Cys-101, while Fe2 is genase (Incharoensakdi and Wutipraditkul, 1999). coordinated by His-83 and His-104. The mononuclear CAO catalyzes the first step in the conversion of iron at the active site is coordinated by the two his- chlorophyll a (Chl a)toChlb (Figure 1c) (Tanaka tidine residues and one carboxylate within the motif et al., 1998). The conservation of this enzyme in the E100-x3-D205-x2-H208x4-H213 and by D362. This prochlorophytes Prochloron didemni and Prochloro- motif is now referred to as a 2-His-1-carboxylate facial thrix hollandica suggests that this enzymatic activity triad (Lange and Que, 1998). Electron transfer from arose once in the common ancestor of oxygenic bac- the Rieske domain to the active site occurs between teria and chloroplasts (Tomitani et al., 1999). The two a subunits which are held in close proximity by absence of a close homologue of CAO from the gen- the hydrogen bonding of D205 to both H104 in the ome of Prochlorococcus which synthesizes divinyl Rieske domain and H208 at the active site (Kauppi Chl b has given rise to the proposition that a Chl b et al., 1998). The conservation of these iron-binding synthase in this organism may have a different phylo- motifs in LLS1-like proteins (Gray et al., 1997, 2002) genetic origin involving convergent evolution (Hess suggests a common reaction mechanism, but not ne- et al., 2001). PTC52 from barley (PORA translocation cessarily common enzyme substrates. Individual mi- complex) is a 52 kDa protein that is associated with the crobial ARH enzymes may operate on several sub- translocation of protochlorophyllide oxidoreductase A strates, for example, vanillate demethylase (VanA) (PORA). The detection of pchlide b within this com- from Acinetobacter, which can act upon the vanil- plex suggests that PTC52 catalyzes the conversion of late analogues m-anisate, m-toluate and 4-hydroxy- pchlide a to pchlide b in a reaction that is analagous to 3,5-dimethylbenzoate (Moraswki et al., 2000). The that of CAO (S. Reinbothe et al., in press). microbial ARH enzymes were originally classified by The recent report that the AtLls1 gene encodes Batie et al.