Information

What is the evolutionary relationship between heme, chlorophyll and other tetrapyrroles?


As a non-biologist, I have searched the Internet and found dozens of papers discussing the similarity between the structures of heme and chlorophyll molecules, but I could not find any discussion of their evolutionary relationship.

To be more specific, I would like to know whether the use of tetrapyrrole in the blood and in photosynthesis can be traced to the earliest organisms in which tetrapyrrole had some function, and then the molecule evolved to be capable of the different functions it currently performs in animals and plants.


Preamble

I found this question interesting, so that, despite my general ignorance in this area, I have attempted to address the question of the evolution of tetrapyrroles rather than answer it. Additional contributions are welcome as comments. I apologize for the large graphic, which I feel is indispensable to my answer, however I have included a summary above it, so readers can decide whether they wish to read further.

Summary

  1. There are four main types of tetrapyrrole associated with proteins
  2. They share a common pathway from 5-aminolevulinic acid to uroporphyrinogen II
  3. The unresolved question of which subsequent pathway evolved first is discussed in relation to their antiquity and complexity.
  4. The possible evolutionary relevance of the two different pathways for the synthesis of 5-aminolevulinic acid - found in different organisms - is also discussed.

Tetrapyrroles in proteins - Diversity and Chemical Rationale

Although the cyclic tetrapyrrole structure looks complex, it appears to be very ancient, presumably because of the greater chemical versatility of its complexes with metal ions in proteins, compared with simpler proteins that contain metal ions. The ring provides a precise four-co-ordination which can be held in position to provide two additional co-ordination sites (at right angles to the plane of the ring) which can interact with protein side-chains or other molecules (such as oxygen). The combination of ion, substitution of the tetrapyrrole and interaction with the protein allows fine-tuning of the redox properties of the ion.

The diagram below (adapted from Zappa et al.) shows the four main types of tetrapyrrole in proteins and how their biosynthesis is related. The two mentioned in the question are actually both derivatives of protoporphyrin IX: chlorins, such as chlorophyll - with its role in photosynthesis - and haem proteins - with the examples of the cytochromes of the electron transport chain being more widespread than that of haemoglobin in eukaryotes. In addition, however, are the corrins - most notably in Vitamin B12 with a role in methyl transfer - and sirohaem - which occurs in certain sulphite and nitrite reductases.

Which tetrapyrrole is oldest in evolutionary terms?

How can we address the question of which tetrapyrrole was first to arise? This question is, in fact, which synthetic pathway - which series of enzymes - was first to arise. In my review of the literature I (like the poster) find this an area which angels are fearful of, but, being foolhardy, I suggest two criteria. The first is what we consider the relative ages of the functions the proteins subserve, and the second the relative complexity of the pathways needed to synthesize them.

The first criterion is subject to dispute. Unfortunately the easy comparison between haem as an oxygen carrier in eukaryotes and chlorophylls as ancient prokaryotic photosynthetic proteins is complicated by the more ancient role of haem in cytochromes. One might argue that cytochromes are a feature of aerobic metabolism that followed the emergence of oxygen generation by photosynthesis, but unfortunately the electron transport chain exists in anaerobic organisms, where the terminal electron acceptor may be nitrate, nitrite, ferric iron, sulphate or carbon dioxide. Given this, and the greater complexity of the pathway from protoporphyrin IX to bacteriochlorophyll a compared with that to protohaem (10 steps compared to one), one might argue that chlorins emerged after haems.

But what about the other tetrapyrroles? One approach to relative ages is to consider the biochemical processes that might have been present early after the emergences of life. One such analysis of this was made by Weiss et al. in a paper published in Nature Microbiology (2016) 1, 1-8, entitled “The physiology and habitat of the last universal common ancestor”. They drew the conclusion that the Last Universal Common Ancestor (LUCA) was an anaerobic autotroph that existed in a hydrothermal setting and obtained energy using a Wood-Ljungdahl pathway with hydrogen as the electron donor and carbon dioxide as the electron acceptor. The two types of tetrapyrroles that they postulate would have been present in the LUCA are corrin (as cobalamin for methyl group transfer) and sirohaem for sulphur metabolism (sulphite reductase) in the hydrothermal environment. No mention of porphyrins! Given the complexity of the 16 step pathway to corrin, compared to the one-step pathway to sirohaem, one might suggest that the latter evolved first.

Synthesis of 5-aminolevulinic acid - evolutionary implications?

5-aminolevulinic acid (frequently referred to by its old name of δ-aminolevulinic acid) is the precursor of uroporphyrinogen II, the common tetrapyrrole precursor. However there exist two pathways by which this can be formed, of which, as I understand it, only one is present in any organism. One of these (at least to my thinking) could have evolutionary implications. These pathways are shown below in a figure adapted from a paper on prokaryotic haem biosynthesis by H. Panek and M.R. O'Brian (2002).

The intriguing aspect of this is that one of the pathways (the C5 pathway) involves reduction of glutamyl-tRNA, whereas the other (the Shemin pathway) is a conventional synthase reaction with substrates glycine and succinyl-CoA. Arguments supporting the RNA world hypothesis include catalytic RNA, and the presence of nucleosides in molecules such as NAD where there complexity belies the simplicity of their function. So, putting my head over the parapet again (to vary my metaphors), I would suggest that the use of glutamyl-tRNA for the synthesis of this ancient and important intermediate might be another fossil of the RNA world. (Perhaps not what the poster had in mind in posing his question.)


Note the molecules are not that similar, only one portion is similar.

The similar portion in each molecule is a cyclic tetrapyrroles. Cyclic tetrapyrroles and porphyrins in particular are a fairly common structure in biology they have several functional uses and are fairly easy to construct from existing simpler biological molecules. The fact that they are functionally good at binding metals makes them a reoccuring solution, although not all Cyclic tetrapyrroles bind metals. Many many biological molecules contain Cyclic tetrapyrroles.

Many biological molecules have similar structures to other molecules, especially ones that perform similar tasks, there are only so many shapes you can make with carbon atoms, the shape of a molecule affects its chemical properties and that shape is simple and has useful properties. Heme itself is a functional component of many proteins called hemoproteins, basically whenever they need to bind iron. Another cyclic tetrapyrroles would be cobalamin otherwise known as vitamin B12 which binds cobalt.

Its the same reason hammer handles look very similar even in very different cultures, just attaching a stick is a simple easy to discover solution, so it is the one that gets found most of the time. Functionally that is the shape that works. Or to put it another way they are only so many shapes you can make a wheel.

https://www.sciencedirect.com/science/article/pii/S0167488912001000

http://www.org-chem.org/yuuki/porphyrin/porphyrin.html

https://www.cpp.edu/~lsstarkey/courses/CHM-Lab/PorphyrinBasics.pdf

https://www.mdpi.com/journal/molecules/special_issues/porphyrin_chemistry

To address the updated question. I honestly don't know if they share an evolutionary origin. But given that they are synthesised in different organelles (mitochondria for heme and chloroplast for chlorophyll, I would guess no.


Patterns in evolutionary origins of heme, chlorophyll a and isopentenyl diphosphate biosynthetic pathways suggest non-photosynthetic periods prior to plastid replacements in dinoflagellates

The ancestral dinoflagellate most likely established a peridinin-containing plastid, which have been inherited in the extant photosynthetic descendants. However, kareniacean dinoflagellates and Lepidodinium species were known to bear “non-canonical” plastids lacking peridinin, which were established through haptophyte and green algal endosymbioses, respectively. For plastid function and maintenance, the aforementioned dinoflagellates were known to use nucleus-encoded proteins vertically inherited from the ancestral dinoflagellates (vertically inherited- or VI-type), and those acquired from non-dinoflagellate organisms (including the endosymbiont). These observations indicated that the proteomes of the non-canonical plastids derived from a haptophyte and a green alga were modified by “exogenous” genes acquired from non-dinoflagellate organisms. However, there was no systematic evaluation addressing how “exogenous” genes reshaped individual metabolic pathways localized in a non-canonical plastid.


Contents

Hemoproteins have diverse biological functions including the transportation of diatomic gases, chemical catalysis, diatomic gas detection, and electron transfer. The heme iron serves as a source or sink of electrons during electron transfer or redox chemistry. In peroxidase reactions, the porphyrin molecule also serves as an electron source, being able to delocalize radical electrons in the conjugated ring. In the transportation or detection of diatomic gases, the gas binds to the heme iron. During the detection of diatomic gases, the binding of the gas ligand to the heme iron induces conformational changes in the surrounding protein. [7] In general, diatomic gases only bind to the reduced heme, as ferrous Fe(II) while most peroxidases cycle between Fe(III) and Fe(IV) and hemeproteins involved in mitochondrial redox, oxidation-reduction, cycle between Fe(II) and Fe(III).

It has been speculated that the original evolutionary function of hemoproteins was electron transfer in primitive sulfur-based photosynthesis pathways in ancestral cyanobacteria-like organisms before the appearance of molecular oxygen. [8]

Hemoproteins achieve their remarkable functional diversity by modifying the environment of the heme macrocycle within the protein matrix. [9] For example, the ability of hemoglobin to effectively deliver oxygen to tissues is due to specific amino acid residues located near the heme molecule. [10] Hemoglobin reversibly binds to oxygen in the lungs when the pH is high, and the carbon dioxide concentration is low. When the situation is reversed (low pH and high carbon dioxide concentrations), hemoglobin will release oxygen into the tissues. This phenomenon, which states that hemoglobin's oxygen binding affinity is inversely proportional to both acidity and concentration of carbon dioxide, is known as the Bohr effect. [11] The molecular mechanism behind this effect is the steric organization of the globin chain a histidine residue, located adjacent to the heme group, becomes positively charged under acidic conditions (which are caused by dissolved CO2 in working muscles, etc.), releasing oxygen from the heme group. [12]

Major hemes Edit

There are several biologically important kinds of heme:

Heme A Heme B Heme C Heme O
PubChem number 7888115 444098 444125 6323367
Chemical formula C49H56O6N4Fe C34H32O4N4Fe C34H36O4N4S2Fe C49H58O5N4Fe
Functional group at C3 –CH(OH)CH2Far –CH=CH2 –CH(cystein-S-yl)CH3 –CH(OH)CH2Far
Functional group at C8 –CH=CH2 –CH=CH2 –CH(cystein-S-yl)CH3 –CH=CH2
Functional group at C18 –CH=O –CH3 –CH3 –CH3

The most common type is heme B other important types include heme A and heme C. Isolated hemes are commonly designated by capital letters while hemes bound to proteins are designated by lower case letters. Cytochrome a refers to the heme A in specific combination with membrane protein forming a portion of cytochrome c oxidase. [15]

Other hemes Edit

  • Heme l is the derivative of heme B which is covalently attached to the protein of lactoperoxidase, eosinophil peroxidase, and thyroid peroxidase. The addition of peroxide with the glutamyl-375 and aspartyl-225 of lactoperoxidase forms ester bonds between these amino acid residues and the heme 1- and 5-methyl groups, respectively. [16] Similar ester bonds with these two methyl groups are thought to form in eosinophil and thyroid peroxidases. Heme l is one important characteristic of animal peroxidases plant peroxidases incorporate heme B. Lactoperoxidase and eosinophil peroxidase are protective enzymes responsible for the destruction of invading bacteria and virus. Thyroid peroxidase is the enzyme catalyzing the biosynthesis of the important thyroid hormones. Because lactoperoxidase destroys invading organisms in the lungs and excrement, it is thought to be an important protective enzyme. [17]
  • Heme m is the derivative of heme B covalently bound at the active site of myeloperoxidase. Heme m contains the two ester bonds at the heme 1- and 5-methyl groups also present in heme l of other mammalian peroxidases, such as lactoperoxidase and eosinophil peroxidase. In addition, a unique sulfonamide ion linkage between the sulfur of a methionyl amino-acid residue and the heme 2-vinyl group is formed, giving this enzyme the unique capability of easily oxidizing chloride and bromide ions to hypochlorite and hypobromite. Myeloperoxidase is present in mammalian neutrophils and is responsible for the destruction of invading bacteria and viral agents. It perhaps synthesizes hypobromite by "mistake". Both hypochlorite and hypobromite are very reactive species responsible for the production of halogenated nucleosides, which are mutagenic compounds. [18][19]
  • Heme D is another derivative of heme B, but in which the propionic acid side chain at the carbon of position 6, which is also hydroxylated, forms a γ-spirolactone. Ring III is also hydroxylated at position 5, in a conformation trans to the new lactone group. [20] Heme D is the site for oxygen reduction to water of many types of bacteria at low oxygen tension. [21]
  • Heme S is related to heme B by having a formal group at position 2 in place of the 2-vinyl group. Heme S is found in the hemoglobin of a few species of marine worms. The correct structures of heme B and heme S were first elucidated by German chemist Hans Fischer. [22]

The names of cytochromes typically (but not always) reflect the kinds of hemes they contain: cytochrome a contains heme A, cytochrome c contains heme C, etc. This convention may have been first introduced with the publication of the structure of heme A.

Use of capital letters to designate the type of heme Edit

The practice of designating hemes with upper case letters was formalized in a footnote in a paper by Puustinen and Wikstrom [23] which explains under which conditions a capital letter should be used: "we prefer the use of capital letters to describe the heme structure as isolated. Lowercase letters may then be freely used for cytochromes and enzymes, as well as to describe individual protein-bound heme groups (for example, cytochrome bc, and aa3 complexes, cytochrome b5, heme c1 of the bc1 complex, heme a3 of the aa3 complex, etc)." In other words, the chemical compound would be designated with a capital letter, but specific instances in structures with lowercase. Thus cytochrome oxidase, which has two A hemes (heme a and heme a3) in its structure, contains two moles of heme A per mole protein. Cytochrome bc1, with hemes bH, bL, and c1, contains heme B and heme C in a 2:1 ratio. The practice seems to have originated in a paper by Caughey and York in which the product of a new isolation procedure for the heme of cytochrome aa3 was designated heme A to differentiate it from previous preparations: "Our product is not identical in all respects with the heme a obtained in solution by other workers by the reduction of the hemin a as isolated previously (2). For this reason, we shall designate our product heme A until the apparent differences can be rationalized.". [24] In a later paper, [25] Caughey's group uses capital letters for isolated heme B and C as well as A.

The enzymatic process that produces heme is properly called porphyrin synthesis, as all the intermediates are tetrapyrroles that are chemically classified as porphyrins. The process is highly conserved across biology. In humans, this pathway serves almost exclusively to form heme. In bacteria, it also produces more complex substances such as cofactor F430 and cobalamin (vitamin B12). [26]

The pathway is initiated by the synthesis of δ-aminolevulinic acid (dALA or δALA) from the amino acid glycine and succinyl-CoA from the citric acid cycle (Krebs cycle). The rate-limiting enzyme responsible for this reaction, ALA synthase, is negatively regulated by glucose and heme concentration. Mechanism of inhibition of ALAs by heme or hemin is by decreasing stability of mRNA synthesis and by decreasing the intake of mRNA in the mitochondria. This mechanism is of therapeutic importance: infusion of heme arginate or hematin and glucose can abort attacks of acute intermittent porphyria in patients with an inborn error of metabolism of this process, by reducing transcription of ALA synthase. [27]

The organs mainly involved in heme synthesis are the liver (in which the rate of synthesis is highly variable, depending on the systemic heme pool) and the bone marrow (in which rate of synthesis of Heme is relatively constant and depends on the production of globin chain), although every cell requires heme to function properly. However, due to its toxic properties, proteins such as Hemopexin (Hx) are required to help maintain physiological stores of iron in order for them to be used in synthesis. [28] Heme is seen as an intermediate molecule in catabolism of hemoglobin in the process of bilirubin metabolism. Defects in various enzymes in synthesis of heme can lead to group of disorder called porphyrias, these include acute intermittent porphyria, congenital erythropoetic porphyria, porphyria cutanea tarda, hereditary coproporphyria, variegate porphyria, erythropoietic protoporphyria. [29] [ citation needed ]

Impossible Foods, producers of plant-based meat substitutes, use an accelerated heme synthesis process involving soybean root leghemoglobin and yeast, adding the resulting heme to items such as meatless (vegan) Impossible burger patties. The DNA for leghemoglobin production was extracted from the soybean root nodules and expressed in yeast cells to overproduce heme for use in the meatless burgers. [30] This process claims to create a meaty flavor in the resulting products. [31] [32]

Degradation begins inside macrophages of the spleen, which remove old and damaged erythrocytes from the circulation. In the first step, heme is converted to biliverdin by the enzyme heme oxygenase (HO). [33] NADPH is used as the reducing agent, molecular oxygen enters the reaction, carbon monoxide (CO) is produced and the iron is released from the molecule as the ferrous ion (Fe 2+ ). [34] CO acts as a cellular messenger and functions in vasodilation. [35]

In addition, heme degradation appears to be an evolutionarily-conserved response to oxidative stress. Briefly, when cells are exposed to free radicals, there is a rapid induction of the expression of the stress-responsive heme oxygenase-1 (HMOX1) isoenzyme that catabolizes heme (see below). [36] The reason why cells must increase exponentially their capability to degrade heme in response to oxidative stress remains unclear but this appears to be part of a cytoprotective response that avoids the deleterious effects of free heme. When large amounts of free heme accumulates, the heme detoxification/degradation systems get overwhelmed, enabling heme to exert its damaging effects. [28]

heme heme oxygenase-1 biliverdin + Fe 2+
H + + NADPH + O2 NADP + + CO

In the second reaction, biliverdin is converted to bilirubin by biliverdin reductase (BVR): [37]

Bilirubin is transported into the liver by facilitated diffusion bound to a protein (serum albumin), where it is conjugated with glucuronic acid to become more water-soluble. The reaction is catalyzed by the enzyme UDP-glucuronosyltransferase. [38]

This form of bilirubin is excreted from the liver in bile. Excretion of bilirubin from liver to biliary canaliculi is an active, energy-dependent and rate-limiting process. The intestinal bacteria deconjugate bilirubin diglucuronide and convert bilirubin to urobilinogens. Some urobilinogen is absorbed by intestinal cells and transported into the kidneys and excreted with urine (urobilin, which is the product of oxidation of urobilinogen, and is responsible for the yellow colour of urine). The remainder travels down the digestive tract and is converted to stercobilinogen. This is oxidized to stercobilin, which is excreted and is responsible for the brown color of feces. [39]


A Cytochrome b Origin of Photosynthetic Reaction Centers: an Evolutionary Link between Respiration and Photosynthesis

The evolutionary origin of photosynthetic reaction centers has long remained elusive. Here, we use sequence and structural analysis to demonstrate an evolutionary link between the cytochrome b subunit of the cytochrome bc1 complex and the core polypeptides of the photosynthetic bacterial reaction center. In particular, we have identified an area of significant sequence similarity between a three contiguous membrane-spanning domain of cytochrome b, which contains binding sites for two hemes, and a three contiguous membrane-spanning domain in the photosynthetic reaction center core subunits, which contains binding sites for cofactors such as (bacterio)chlorophylls, (bacterio)pheophytin and a non-heme iron. Three of the four heme ligands in cytochrome b are found to be conserved with the cofactor ligands in the reaction center polypeptides. Since cytochrome b and reaction center polypeptides both bind tetrapyrroles and quinones for electron transfer, the observed sequence, functional and structural similarities can best be explained with the assumption of a common evolutionary origin. Statistical analysis further supports a distant but significant homologous relationship. On the basis of previous evolutionary analyses that established a scenario that respiration evolved prior to photosynthesis, we consider it likely that cytochrome b is the evolutionary precursor for type II reaction center apoproteins. With a structural analysis confirming a common evolutionary origin of both type I and type II reaction centers, we further propose a novel “reaction center apoprotein early” hypothesis to account for the development of photosynthetic reaction center holoproteins.


Conclusions

The tetrapyrrole biosynthesis pathway in phototrophic eukaryotes is an evolutionary mosaic originating in proteobacteria, cyanobacteria and eukaryotes. It represents a shopping bag of enzymes collected during the history of plastid endosymbiosis retained, due to its essential role in metabolism, even after photosynthetic capabilities have been lost. Here we confirm that the tertiary plastids of dinotoms represent largely independent compartments with tetrapyrrole biosynthesis occurring parallel to biosynthesis in the peridinin plastid. The enzymes putatively localized to the former plastid branch sister to dinoflagellate enzymes, while the tertiary plastid contains enzymes branching sister to those of diatoms, mirroring the origin of the respective organelles. In G. theta, the pathway is located almost entirely in the plastid, with the exception of a eukaryotic ferrochelatase apparently localized to the mitochondrion, indicating either a slow evolutionary rate or an evolutionary constraint. Furthermore, we observed that the majority of the pathway is evolutionarily conserved and related to the red lineage even in organisms that currently possess a plastid of green algal provenance, i.e. the dinoflagellate Lepidodinium chlorophorum and the chlorarachniophyte B. natans. Hence, if the protein targeting machinery is compatible with the new plastid compartment, the tetrapyrrole synthesis pathway can be relocated “as is”, which is illustrated in the case of L. chlorophorum. Intriguingly, such a scenario may imply the existence of a cryptic red-derived plastid earlier in the history of chlorarachniophytes. While the evolution of eukaryotes is becoming clearer with increasing data from deeper lineages, the history of plastid acquisitions resists revealing an unequivocal scenario due to massive gene transfer and phylogenetic bias. We suggest that a targeted approach directed at conserved processes could result in new, relevant hypotheses even in the genomic era.


METHODS

Sequence Retrieval and Phylogenetic Analyses

Partial sequences of the genes of the heme biosynthesis pathway were searched in the sequence data of the 454 genome sequencing surveys of Chromera velia and CCMP3155 ( Janouškovec et al., 2010) by BLAST 2.2.18 (http://blast.ncbi.nlm.nih.gov). Complete coding sequences were amplified from the cDNA of C. velia by 3′ RACE and 5′ RACE using the FirstChoice RLM-RACE kit (Ambion), cloned, and sequenced. For each enzyme, appropriate homologs were identified by BLAST and downloaded from Web sources (see Supplemental Table 1 online ), aligned using MAFFT ( Katoh and Toh, 2008), manually edited using BioEdit ( Hall, 1999), and used for further phylogenetic analyses (see Supplemental Data Sets 1 to 3 online ). Bayesian trees were computed using PhyloBayes 3.2d under CAT-GTR evolutionary model ( Lartillot and Philippe, 2004). For each analysis, two independent chains were run for a sufficient number of steps after the chains converged. The convergence of two independent chains was assessed based on bpcomp analysis (PhyloBayes 3.2d). The burnin value was chosen according to the same analysis (usually 20% of the trees were discarded). It should be noted that the presence of Plasmodium sequences in our analyses did not support any other method of tree construction because of their high divergence. Only the use of the advanced CAT model supported the expected placement of Plasmodium into the same clade with other apicomplexans in many of the analyses. Thus, the trees show only Bayesian posterior probabilities and not any other support, such as maximum likelihood bootstraps. We preferred to use all apicomplexan sequences, including those that are highly divergent, before the use of other additional phylogenetic methods on the reduced data set.

Putative Localizations of Enzymes of the Tetrapyrrole Synthesis in C. velia

Putative localizations of nuclear encoded enzymes of the tetrapyrrole pathway were tested by in silico predictions using CBS prediction servers (http://www.cbs.dtu.dk/services/). In particular, programs SignalP and TargetP ( Emanuelsson et al., 2007) were used to predict SPs and TPs, respectively.

In Vivo Radiolabeling of Chlorophyll

To perform in vivo radiolabeling of chlorophyll, 50 mL of C. velia or Synechocystis 6803 cells at OD750

0.4 were concentrated into 350 μL of growth media supplemented with 20 mM TES/NaOH buffer, pH 8.0, transferred into a glass tube, and incubated on a shaker for 1 h at 30°C and at 80 μmol of photons s −1 m −2 . Then, 350 μM 14 C-Glu or 14 C-Gly was added (specific activity

50 mCi/mmol American Radiolabeled Chemicals), and cells were further incubated under the same conditions. Incorporation of labeled amino acid into cells was monitored by measuring decrease of the radioactivity from the supernatant every 30 min on liquid scintillation analyzer (Packard Tri-Carb 1500). After 2 h, cells were washed three times with water and then pigments were extracted from pelleted cells using 1.3 mL of methanol/0.2% NH4. This solution was mixed with 140 μL of 1 M NaCl and 400 μL of hexane and vortexed. Hexane phase containing chlorophyll was removed and evaporated on a vacuum concentrator, resuspended in 100 μL of methanol, and mixed with 100 μL of methanol containing 10% KOH. After incubation for 15 min at room temperature, the solution was extracted twice with 200 μL hexane and once by 200 μL of petroleum ether (boiling range 40 to 65°C) to remove phytol and unprocessed chlorophyll, and finally acidified by 20 μL of concentrated HCl. The resulting solution containing the chlorophyll derivate chlorin was cleared by centrifugation, the supernatant transferred into a new tube, dried on a vacuum concentrator, and resuspended in a drop of methanol. Reverse-phase thin layer chromatography was performed on silica-gel plate (Macherey-Nagel) chloroform:methanol:water (65:25:3) was used as the mobile phase. After separation, the plate was dried and the signal captured on an x-ray film (exposure time of 5 d).

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: HQ222925 to HQ222935 for the complete genes of the tetrapyrrole biosynthesis of C. velia and HQ245653 to HQ245656 for the partial gene sequences of the strain CCMP3155. Accession numbers for sequences used in phylogenetic analysis are presented in Supplemental Table 1 online .

Supplemental Data

Supplemental Figure 1. Phylogenetic Trees of Δ-Aminolevulinic Acid Dehydratase, Porphobilinogen Deaminase, Uroporphyrinogen Synthase, Coproporphyrinogen Oxidase, and Protoporphyrinogen Oxidase.

Supplemental Figure 2. Comparison of ALAS N-Terminal Presequences of Various Eukaryotes, Including C. velia.

Supplemental Figure 3. Presence of Conserved Heme Regulatory Motifs (CP Motifs) in the N-Terminal Presequences of Δ-Aminolevulinic Acid Synthases of Various Eukaryotes, Including C. velia.

Supplemental Table 1. Sequences Used in the Analyses.

Supplemental Data Set 1 . Text File of Alignment Corresponding to Figure 1.

Supplemental Data Set 2 . Text File of Alignment Corresponding to Figure 2.

Supplemental Data Set 3 . Text File of Alignment Corresponding to Supplemental Figure 1.


Materials and Methods

Sequence Retrieval and Phylogenetic Analyses

Sequences of the genes of the heme biosynthesis pathway were searched in the available sequence databases: PEPdb (http://amoebidia.bcm.umontreal.ca/pepdb), GenBank and E. gracilis genome data were obtained from the MC Field laboratory sequence database at: http://web.me.com/mfield/Euglena_gracilis (see supplementary table S2 , Supplementary Material online for sequence IDs). Where only small pieces of genes available, we amplified larger gene segments from the cDNA of E. gracilis strain Z (kindly provided by Professor Krajčovič, Bratislava). The 5' regions were amplified using the FirstChoice RLM-RACE kit (Ambion) or the spliced-leader specific primer (5'-ACACTTTCTGAGTGTCTATTTTTTTTCG-3'). All newly sequenced data were deposited in GenBank under accession numbers: JF292577-JF292587.

For each enzyme, appropriate homologues were identified by Blast, aligned using MAFFT ( Katoh and Toh 2008) and manually edited using BioEdit ( Hall 1999). Phylogenetic trees were computed using PhyloBayes 3.2f under the CAT-GTR evolutionary model ( Lartillot and Philippe 2004). For each analysis, two independent chains were run for at least 50,000 steps. The convergence of the chains was assessed based on bpcomp analysis (PhyloBayes 3.2f). For the 50% majority consensus trees, each 10th tree was sampled and the first 10% of the trees were discarded. The bootstrap support values were calculated in RAxML 7.0.3 ( Stamatakis 2006) using the PROTGAMMALG model after 1,000 replications.


Materials and Methods

Sequences

All homologs of HemY, HemG, and HemJ were retrieved from the Gclust database (Gclust2010e29b data set at http://gclust.c.u-tokyo.ac.jp/ [last accessed August 12, 2014, now available under “Old versions”] including selected genomes covering plants, algae, nonphotosynthetic eukaryotes, and prokaryotes.) according to the published list of homologs. Gclust is a comparative genomic database of homologous protein clusters suitable for phylogenetic profiling ( Sato 2002, 2009 Sato et al. 2005). The sources of the original databases are described on the website.

We identified all homologs of enzymes involved in heme biosynthesis in all prokaryotic genomes in RefSeq of the National Center for Biotechnology Information (NCBI) database as of November 2010 (data set AllBact2010) and used them for the statistics shown in table 2. In most analyses, the two data sets were sufficient for obtaining an idea of the overall distribution of enzymes in various phyla. However, some new sequences were retrieved from RefSeq as of February 2013 to complement the data in detailed analyses for supplementary figures S4 and Supplementary Data , Supplementary Material online. All 3,916,828 proteins in the 1,196 prokaryotic genomes were used in all-against-all BLASTP (mostly v2.2.22) analysis (options: –m8 –FF –C0). The results in a single table (specified by option –m8) were used for clustering by use of gclust v3.56 (parallel version) ( Sato 2009). Each of the enzymes in the heme biosynthetic pathway was first identified by name, then all homologs were retrieved as several clusters. Additionally, some singletons were retrieved by following the “related groups” link. A list of enzymes in all analyzed organisms was compiled from the lists of homologs. The huge file of original data of clusters (7.12 GB) is not currently available on the web site but may be obtained from the corresponding author upon request.

Phylogenetic Analyses

Amino acid alignment was performed for each isofunctional enzymes by using Muscle v3.6 ( Edgar 2004). Sequences not aligned in the entire length were removed, and alignment was repeated. The sequence alignment was used for subsequent phylogenetic analysis and homology modeling. The sites with gaps in more than 20% of sequences were removed by use of SISEQ v1.59 ( Sato 2000), which was also used for conversion of various sequence formats. ClustalX v1.83 was used for profile alignment and to manage aligned sequences ( Thompson et al. 1994).

Each alignment was used in the phylogenetic analysis as follows. A neighbor-joining (NJ) tree was estimated with MEGA v4 ( Tamura et al. 2007) with the Jones–Taylor–Thorton (JTT) model and an equal evolutionary rate. Calculation with the ML method involved use of TreeFinder March 2008 version ( Jobb et al. 2004) with the Whelan–Goldman (WAG) model, and with RAxML v7.0.4 ( Stamatakis 2006) with –f d –i 10 –m PROTCATWAG options. The exact parameters were determined by initial trials with –c 10, 40, 55 with or without –i 10. Bootstrap was based on 1,000 replicates. Bayesian inference (BI) involved use of MrBayes v3.2 ( Ronquist and Huelsenbeck 2003), with the following options: aamodelpr = fixed(wag), ratepr = variable, ngen = 2,000,000, samplefreq = 200, burnin = 3,000 (for HemG, ngen = 1,000,000, samplefreq = 100), and with PhyloBayes v3.2e ( Lartillot et al. 2009) with the CAT+gtr model. A 16S–23S-based phylogenetic tree in Cyanobacteria ( fig. 2) was constructed with the BI method as described ( Sasaki and Sato 2010).

The approximately unbiased (AU) test, intended to test the relative likelihood of various forms of trees based on support levels of individual sites, involved use of the CONSEL program ( Shimodaira and Hasegawa 2001), with the output of Protml in MOLPHY v2.3beta ( Adachi and Hasegawa 1996), based on the 19 most probable trees for 20 representative taxa. For this purpose, the trees were selected by Protml with constrained trees according to the results of ML and BI analyses. The WAG and JTT models were used. Phylogenetic trees were drawn with use of NJplot ( Perrière and Gouy 1996) and Mesquite utility v2.5 (http://mesquiteproject.org, last accessed August 12, 2014). The alignment files used for phylogenetic analysis are available in supplementary material , Supplementary Material online.

Homology Modeling

Homology modeling of HemY homologues involved use of Modeller v8v1 or 8v2 ( Sali and Blundell 1993) with the automodel script, modified as necessary. Both of the structures in the Protein Data Bank (PDB) entry 1SEZ for tobacco Protox (PPO2) were used as templates. This file describes two monomers arranged in a symmetric position, but some loops and the N-terminus are invisible because of high flexibility. The corresponding parts in the inferred models are an invention of the software and are only meaningful as an indication of the presence of a structural part. Cartoon models of structure were prepared with use of Molscript ( Kraulis 1991). The coordinate files of homology modeling are available from the corresponding author upon request.


Conclusion

In summary, HrHEMA, HrPOR and HrCAO silencing can cause leaf yellowing and chloroplast structure changes in Hosta. On note, leaves of Hosta with HrCAO silencing were the most affected, while the HrPOR silencing plant was the least affected. The overexpression of these three genes in tobacco enhanced photosynthesis by accumulating chlorophyll content, but the influential level varied under different light intensities. Furthermore, HrHEMA-, HrPOR- and HrCAO- overexpressing in tobacco can enhance the antioxidant capacity of plants to cope with stress under higher light intensity. However, under lower light intensity, the antioxidant capacity deteriorated in the HrHEMA-, HrPOR- and HrCAO-overexpressing tobaccos.


METHODS

Sequence Retrieval and Phylogenetic Analyses

Partial sequences of the genes of the heme biosynthesis pathway were searched in the sequence data of the 454 genome sequencing surveys of Chromera velia and CCMP3155 (Janouškovec et al., 2010) by BLAST 2.2.18 (http://blast.ncbi.nlm.nih.gov). Complete coding sequences were amplified from the cDNA of C. velia by 3′ RACE and 5′ RACE using the FirstChoice RLM-RACE kit (Ambion), cloned, and sequenced. For each enzyme, appropriate homologs were identified by BLAST and downloaded from Web sources (see Supplemental Table 1 online), aligned using MAFFT (Katoh and Toh, 2008), manually edited using BioEdit (Hall, 1999), and used for further phylogenetic analyses (see Supplemental Data Sets 1 to 3 online). Bayesian trees were computed using PhyloBayes 3.2d under CAT-GTR evolutionary model (Lartillot and Philippe, 2004). For each analysis, two independent chains were run for a sufficient number of steps after the chains converged. The convergence of two independent chains was assessed based on bpcomp analysis (PhyloBayes 3.2d). The burnin value was chosen according to the same analysis (usually 20% of the trees were discarded). It should be noted that the presence of Plasmodium sequences in our analyses did not support any other method of tree construction because of their high divergence. Only the use of the advanced CAT model supported the expected placement of Plasmodium into the same clade with other apicomplexans in many of the analyses. Thus, the trees show only Bayesian posterior probabilities and not any other support, such as maximum likelihood bootstraps. We preferred to use all apicomplexan sequences, including those that are highly divergent, before the use of other additional phylogenetic methods on the reduced data set.

Putative Localizations of Enzymes of the Tetrapyrrole Synthesis in C. velia

Putative localizations of nuclear encoded enzymes of the tetrapyrrole pathway were tested by in silico predictions using CBS prediction servers (http://www.cbs.dtu.dk/services/). In particular, programs SignalP and TargetP (Emanuelsson et al., 2007) were used to predict SPs and TPs, respectively.

In Vivo Radiolabeling of Chlorophyll

To perform in vivo radiolabeling of chlorophyll, 50 mL of C. velia or Synechocystis 6803 cells at OD750

0.4 were concentrated into 350 μL of growth media supplemented with 20 mM TES/NaOH buffer, pH 8.0, transferred into a glass tube, and incubated on a shaker for 1 h at 30ଌ and at 80 μmol of photons s 𢄡 m 𢄢 . Then, 350 μM 14 C-Glu or 14 C-Gly was added (specific activity

50 mCi/mmol American Radiolabeled Chemicals), and cells were further incubated under the same conditions. Incorporation of labeled amino acid into cells was monitored by measuring decrease of the radioactivity from the supernatant every 30 min on liquid scintillation analyzer (Packard Tri-Carb 1500). After 2 h, cells were washed three times with water and then pigments were extracted from pelleted cells using 1.3 mL of methanol/0.2% NH4. This solution was mixed with 140 μL of 1 M NaCl and 400 μL of hexane and vortexed. Hexane phase containing chlorophyll was removed and evaporated on a vacuum concentrator, resuspended in 100 μL of methanol, and mixed with 100 μL of methanol containing 10% KOH. After incubation for 15 min at room temperature, the solution was extracted twice with 200 μL hexane and once by 200 μL of petroleum ether (boiling range 40 to 65ଌ) to remove phytol and unprocessed chlorophyll, and finally acidified by 20 μL of concentrated HCl. The resulting solution containing the chlorophyll derivate chlorin was cleared by centrifugation, the supernatant transferred into a new tube, dried on a vacuum concentrator, and resuspended in a drop of methanol. Reverse-phase thin layer chromatography was performed on silica-gel plate (Macherey-Nagel) chloroform:methanol:water (65:25:3) was used as the mobile phase. After separation, the plate was dried and the signal captured on an x-ray film (exposure time of 5 d).

Accession Numbers

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Phylogenetic Trees of δ-Aminolevulinic Acid Dehydratase, Porphobilinogen Deaminase, Uroporphyrinogen Synthase, Coproporphyrinogen Oxidase, and Protoporphyrinogen Oxidase.

Supplemental Figure 2. Comparison of ALAS N-Terminal Presequences of Various Eukaryotes, Including C. velia.

Supplemental Figure 3. Presence of Conserved Heme Regulatory Motifs (CP Motifs) in the N-Terminal Presequences of δ-Aminolevulinic Acid Synthases of Various Eukaryotes, Including C. velia.

Supplemental Table 1. Sequences Used in the Analyses.

Supplemental Data Set 1. Text File of Alignment Corresponding to Figure 1 .

Supplemental Data Set 2. Text File of Alignment Corresponding to Figure 2 .

Supplemental Data Set 3. Text File of Alignment Corresponding to Supplemental Figure 1.


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