Analyzing the IPP Pathway in Three Labyrinthulomycetes
Daniel Conroy
July 18th, 2014
Introduction
Labyrinthulomycetes are a rarely studied group of osmoheterotrophs, which play a
critical role at the base of the food chain, as well in recycling of nutrients (Raghukumar and
Damare, 2011). Although they are widespread, the chemical and ecological role of
labyrinthulomycetes remains unclear (Raghukumar, 2002). However, there are recent
investigations seeking to utilize the high fatty acid content of these organisms for biofuel and
Omega-3 feedstocks (Chang et al., 2014). Carotenoids are involved in various functions
throughout photosynthetic cells, such as the closest relatives of Labyrinthulomycetes, diatoms.
These organisms use carotenoids in to disperse excess photons in the xanthophyll cycle
(Bertrand, 2010). Isopentenyl Pyrophosphate (IPP) is the precursor of carotenoids, but is
present in all organisms, making its pathway ideal for studying phylogeny as well as the
chemical processes of these cells. The following study investigated the production of IPP in
order to understand weather Labyrinthulomycetes possess the MEP or MVA pathway, and the
phylogeny of the enzymes used in the pathway.
Materials and Methods
The genomes of Aplanochytrium limacinum PBS07, Aurantiochytrium limacinum ATCC
MYA-1381, and Schizochytrium aggregatum ATCC 28209 were searched through the Joint
Genome Institute (http://genome.jgi-psf.org/). The EC or each protein in the MVA and MEP
pathway was searched, and each result was collected and further analyzed. The BLAST program
from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) was
used to find similar sequences. BioEdit was used to align protein sequences as well as to delete
extraneous amino acids. This was also used to divide gene 97214 of Aplanochytrium into three
parts. Phylogenic trees were run using Mega, set as the bootstrap method with 100 replications
and pairwise deletion.
Finding the Possible Genes Involved in IPP Synthesis
To isolate the gene sequences relating each protein involved in the Mevalonate (MVA)
and/or 1-deoxy-D-xylulose 5-phosphate (MEP) pathway, the Enzyme Commission (EC) number
for each enzyme in the pathway was searched in each organism’s genome on JGI.org. For the
enzymes in the MVA pathway, the most searches yielded multiple genes. However, in searching
for enzymes in the MEP pathway, no genes were found. From this we concluded that only the
MVA pathway is present in Labyrinthulomycetes.
Elucidating False Positive Proteins in the MVA Pathway
MPDC Genes
MVA
MPDC
Aplano Auran
Schizo
97214
46997
85181
FIG. 1 MPDC Genes
Proteins found by searching EC 4.1.1.33
for MPDC in each genome on JGI.org
We worked upstream in the MVA Pathway, first analyzing Diphospho-MVA
decarboxylase (MPDC) genes. Although the genome search yielded only one protein for each
organism (Fig.1), further analyses was required. Each MPDC was run through BLAST, and the
100 most similar sequences were copied to BioEdit. Interestingly, the MPDC of Aplanochytrium
was in the same reading frame as a MVA Kinase (MK) and a Phospho-MVA Kinase (PMK), the
enzymes functioning immediately before MPDC in the MVA pathway. To isolate the MPDC
region of Ap97214, the first 300 and last 120 amino acids were removed in Bio Edit, and the
BLAST sequences from the remaining region were taken. The Phylogenic Tree created using
these proteins and those found through BLAST showed that Ap97214 is distantly related to
bacteria MPDCs. Emiliania Huxleyi MPDC is the closest relative of Au46997 and Sc85181, which
also fall near Oomycetes in the tree.
MK and PMK Genes
MVA
Aplano Auran
Schizo
MK/PMK
83434
142637
82155
33802
2444
48997
92651
4319
80502
37505
44216
27367
97214
46997
87938
96378(MK)
43850
85181
125370
98402
76700
41503
117049
Fig.2 MK/PMK Genes
Genes found by searching EC
2.7.1.36/2.7.4.2 for MK/PMK. All results
for this search were the same for both
proteins, except Ap96378, which was
found only under the MK search.
The MVA Kinase (MK) and Phospho-MVA Kinase (PMK) searches on JGI.org yielded
identical results for Aurantiochytrium and Schizochytrium, and had only one difference for
Aplanochytrium (Fig. 2). Most of these genes were annotated in the GHMP Superfamily of
Kinases, but not specifically as PMK or MK. The first step in finding PMK and MK specifically was
to group the genes by similarity using the phylogenic tree and similar amino acid sites. Once
tentative groups were formed, a phylogenic tree including known PMK’s was created, but the
bootstrap support among the branches including Labyrinthulomycetes was too low for
conclusions.
Previous work differentiating kinases offered amino acid sites specific to each type of
GHMP (Andreasssi and Leyh, 2004). By manually searching for the 5-10 amino acid sequences
throughout the 22 genes, and comparing the hits on BLAST, each group was classified as either
a PMK, MK, Galacto Kinase (GLK), Homoserine Kinase (HMK), Mevalonate Decarboxylase
(MVD), or unknown (Fig. 3).
The genes classified as unknown were clearly not an MK or PMK. Ap98378, Au2444 and
Sc48997 showed closer resemblance to glucurono Kinases, although many of their BLAST
sequences were predicted proteins. Ap37502, Au125370, and Sc27367 likely contained a
domain in the GHMP superfamily, but were connected to a glycotransferase, making them
unlikely candidates for involvement in the MVA pathway. Au4319 and Sc87938 resemble
galacto Kinases, and Ap83434 resembled L-fucose Kinase.
Gene 97214 of Aplanochytrium was found to contain three different domains. The first
domain is an MK about 300 Amino Acids long, the second is an MPDC about 400 amino acids
long, and the third domain is a PMK about 185 amino acids long. Each of these domains was
separated on BioEdit to be run through BLAST. Gene 117049 of Schizochytrium is a manually
annotated, extended version of 41503, and includes the conserved regions at the end of the
protein.
Group
Aplano
Auran
Schizo
MK
PMK
HMK
GLK
MVD
97214First 97214End
33802
92651
142637
76700
44216
43850
46997
82155
117049
98402
80502
85181
Aplano
Auran
Schizo
Unknown Unknown Unknown
98378
37505
83434
125370
4319
48997
87938
27637
Fig. 3 Specified Kinases
Proteins were annotated using BLAST results and conserved sites identified
by Andreasssi and Leyh (2004) . Conserved Sites were: GLSSSASLE, GIMDQ,
AE, RARH, and TGGFGGC of Galacto Kinase; TSANL, GLGSSSA, VAGI, and
HPDNVAP of Homoserine Kinase; SLSV, GSGSASRSLYGGY, and CCYTFDAGPN
of Mevalonate Decarboxylase.
A second PMK phylogenic tree containing only Ap97214End, Au76700, Sc117049 and
their BLAST sequences still showed low boot strap values. Further analysis into the 3 different
PMK’s revealed that the Ap97214End resembled an Animal Type PMK, whereas Au76700 and
Sc117049 both resembled the ERG8 Type PMK. To solve this, just Ap97214End and its BLAST
sequences were put into a phylogenic tree. Still, it had low bootstrap values, so more
heterokont sequences found on BLAST were added to the tree. Similarly, Ap97214End was
distantly branched from most sequences in the tree, however it did match well with the PMK of
Acanthoeba castellanii. A separate phylogenic tree containing PMK’s Au76700 and Sc117049
showed that these two enzymes group well together, but poorly with other ERG8 PMK’s.
A new MK Tree created using Ap97214First Domain, Au142637 and Sc82155, and their
BLAST sequences showed the common pattern in which the two Thraustochytrids,
Aurantiochytium and Schizochytrium, align well with each other, whereas Aplanochytrium is
separate, in this case grouped closes to Trichomonas Vaginalis.
HMGR Genes
MVA
HMGR
Aplano Auran
Schizo
47734
141519
81525
46175
124781
85121
103958
47106
70078
40917
98242
49883
78526
86957
Fig.4 HMGR Genes
Genes found by searching 1.1.1.34 in
each genome on JGI.org
Sc86957 and Sc78526 were rejected first because they are too long and contain various
other domains. Upon further analysis, it was clear that most of these genes contained only a
fragment, approximately 20 amino acids long, resembling 3-Hydroxy-3-methylglutaryl-CoAReductase (HMGR). This applied to Sc98242, Sc70078, Ap49883, Au47106, Ap103958 and
Ap40917. Ap49886 was more likely a signal recognition particle, and Ap40917 was more likely a
Mitochondrial Translocase.
The tree containing the remaining 6 possible enzymes, and the BLAST sequences of
Ap47734 and Au124781, showed that Labyrinthulomycete HMGR genes grouped well together.
However, they had low bootstrap support with the other organisms, their closest relative being
Emiliania huxleyi.
After closer examination, each of the 6 HMGRs were Class I and matched very well with
other HMGR genes in the National Center for Biotechnology Information. HMGR Class I genes in
general are well conserved, and differ greatly from Class II HMGRs. In another attempt to
elucidate false HMGRs, the common promoters DAMGMNM and CITMMEA were searched. The
former is present in all 6 proteins, as well as a sequence differing slightly from the latter. This
information evidences that all 6 proteins are HMGRs. Although we do not know of multiple
occurrences of HMGRs in Heterokonts, 3 HMGRs have been reported in Camptotheca
acuminate (Maldonado et al., 1997). However, they are localized in various regions of the plant,
such as the bark, and are evolutionarily beneficial in producing varying amounts of carotenoids
based on different expression levels (Maldonado et al., 1997).
HMGS Genes
The only results when searching JGI.org for 3-Hydroxy-3-methylglutarl-CoA synthase
(HMGS) were gene 48616 for Aurantiochytrium, gene 84768 for Schizochytrium, and
Aplanochtrium had 2 possible genes: 33946 and 46178. A tree formed using these enzymes and
their BLAST sequences showed that Ap33946 grouped well with Au48616 and Sc84768,
although these three genes were distant from the rest of the tree. Ap46178 grouped distantly
to Monosiga brevicollis, as well as Acanthamoeba castellani (with poor bootstrap support).
AACT Genes
MVA
AACT
Aplano Aurantio Schizo
89275
46117
17864
29892
123015
50310
29234
137176
84115
31757
41801
84838
41448
126249
41359
47983
42885
69181
46925
45444
82524
52114
119534
82042
55091
141377
83881
70610
138485
92911
105614
48862
35974
Fig. 5 Acetyl CoA Acetyltransferase Genes
Genes found by searching 2.3.1.9 in each
genome on JGI.org
Each of the 3 Labyrinthulomycetes contained a multitude of possible Acetyl-CoA Cacetyltransferase (AACT) genes. The possible genes was very similar in length, and had large
domains resembling AACT enzymes. For a first attempt at a phylogenic tree, two possible
enzymes from each organism were blasted, and a tree was created using all of them. However,
this was unsuccessful as there was too much variation in the BLAST sequences. There are two
important sites ubiquitous in AACTs, which separate them from similar proteins. CGSGL around
amino acid 89 and CIGGG around amino acid 380 are indicators of AACTs. Both CGSGL and
GIGGG were present, at their respective locations, in Ap89275, Au137176 and Sc50310. CIGGG
was present at the correct location in Ap46925, Au45444, and Sc41359. However, the BLAST
results of Au45444, Ap46925 and Sc41359 showed that each of these proteins more closely
resembled ketoacyl thiolases. By this evidence we concluded that Ap89275, Au137176, and
Sc50310 were the real AACTs active in the MVA pathway.
The tree created using Ap89275, Au137176, and Sc50310 grouped these 3 proteins
together with reasonable bootstrap support. On the other hand their closest relative
Piscirickettsia salmonis was linked to them with a bootstrap value of only 37. The AACT tree
showed four separate groups of heterokonts. No sample from each of the 4 groups possessed
the CGSGL and CIGGG conserved sites of HMGRs. Although the BLAST results from these
samples did, in some cases, show more ketoacyl thiolases, it was not conclusive enough to
adjust the tree accordingly.
Conclusion
The IPP pathway is a useful tool in analyzing the phylogenic relationships as it is
ubiquitous. The protein sequences of these three Labyrinthulomycetes show strong divergence
from others available on BLAST. However, some repetition in neighboring branches was shown.
The complete gene of 97214 from Aplanochytrium is composed a MK, MPDC, and PMK.
This gene is unique, and no protein with even two of these fused domains was found threw
BLAST on NCBI. Furthermore, the PMK residing in the last 180 amino acids of the gene is of the
animal type, whereas the thraustochytrids possess the ERG8 type PMK. This is evidence that
Aplanochytrium first received a PMK through lateral gene transfer. In the Aplanochytrium PMK
tree, this organism grouped only with Acanthamoeba castellanii, but their sequences differed
too greatly to conclude lateral gene transfer. However, the differences in the 97214 sequence
could be due to its combination with the MK and MPDC, which may negate the need for some
substrate binding sites highly conserved in other PMKs. Besides allowing substrate channeling,
the 3 fused domains would allow for more rapid and efficient changes in expression levels. This
would be greatly beneficial, as the xanthophyll cycle reacts to the rapid changes of light
intensity in the water column (Bertrand, 2010). More carotenoids available to undergo
epoxidation would be extremely helpful in short term adaptations.
The MK and MPDC of the Thraustochytrids Aurantiochytrium and Schizochytrium also
branched far from those of Aplanochytrium. The Thraustochytrid MKs grouped with other
heterokonts including Emiliania huxleyi, whereas Aplanochytrium branched closer to
Trichomonas vaginalis. Similarly, in the MPDC tree, Aurantiochytrium and Schizochytrium
grouped with Emiliania huxleyi and other heterokonts, while Aplanochytrium was completely
isolated. This is further evidence that Aplanochytrium has undergone unique mutations or
lateral gene transfer much different from the other two Labyrinthulomycetes.
Further upstream in the pathway, the Labyrinthulomycete genes have evolved much
more similarly. In the HMGR, HMGS, and AACT tree, each of the Labyrinthulomycete genes
grouped together. For the HMGR tree, this was with the heterokonts, but for the HMGS and
AACT trees, Labyrinthulomycete genes were separate, with one or no other organisms. The
presence of 2 HMGR genes in each organism is surprising and requires further research. This
may evidence that carotenoid biosynthesis rates are differentiated in different parts of the cell.
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