Comments

Follow up (dry / wet) in establishing of paaA’s associative pathways and role in communal metabolism would be a significant contribution . I would like to see this reflected/updated in the thesis and/or defence

library(MetamapsDB)
library(dplyr)
library(purrr)
library(igraph)
library(ggplot2)
library(plotly)
library(magrittr)

# MetamapsDB::connect("172.18.0.2", pass="password")
# Please set this when you first initialise the password
paaA = "K02609"
koname(paaA) %>% as_tibble %>% kable

ko.ko	ko.name	ko.definition
ko:K02609	paaA	ring-1,2-phenylacetyl-CoA epoxidase subunit PaaA [EC:1.14.13.149]

KEGG Ortholog ID for ring-1,2-phenylacetyl-CoA epoxidase subunit is: K02609.

ko2path(paaA) %>%
    rowwise %>%
    mutate(num_ko = path2ko(p.pathway) %>% nrow) %>%
    select(p.pathway:num_ko) %>% as_tibble %>% kable

p.pathway	p.pathwayname	num_ko
path:ko00360	Phenylalanine metabolism	75
path:ko01120	Microbial metabolism in diverse environments	1040

Associated Pathways: Phenylalanin Metabolism

Althought paaA belongs in KEGG’s Microbial Metabolism in diverse environments pathway, we will ignore this pathway for now as it has 1040 KOs associated with it.

Below are KOs found in: Phenylalanin metabolism

assoc_kos_definition = path2ko('path:ko00360') %>% map_df(koname)
assoc_kos_definition %<>% setNames(c("ko", "name", "definition"))
# knitr::kable(assoc_kos_definition)
assoc_kos = path2ko('path:ko00360') %>% pull(KO)
assoc_kos_definition %>% as_tibble %>% kable

ko	name	definition
ko:K18852	HPA3	D-amino-acid N-acetyltransferase [EC:2.3.1.36]
ko:K18606	HPPR	hydroxyphenylpyruvate reductase [EC:1.1.1.237]
ko:K18383	K18383	trans-feruloyl-CoA hydratase / vanillin synthase [EC:4.2.1.101 4.1.2.41]
ko:K18363	padD	phenylacetyl-CoA:acceptor oxidoreductase 26-kDa subunit
ko:K18362	padC	phenylacetyl-CoA:acceptor oxidoreductase 27-kDa subunit
ko:K18361	padB	phenylacetyl-CoA:acceptor oxidoreductase [EC:1.17.5.1 3.1.2.25]
ko:K18360	padA	phenylacetyl-CoA:acceptor oxidoreductase accessory protein
ko:K18359	padH	phenylglyoxylate dehydrogenase epsilon subunit [EC:1.2.1.58]
ko:K18358	padF	phenylglyoxylate dehydrogenase delta subunit [EC:1.2.1.58]
ko:K18357	padE	phenylglyoxylate dehydrogenase gamma subunit [EC:1.2.1.58]
ko:K18356	padI	phenylglyoxylate dehydrogenase beta subunit [EC:1.2.1.58]
ko:K18355	padG	phenylglyoxylate dehydrogenase alpha subunit [EC:1.2.1.58]
ko:K15866	paaG	2-(1,2-epoxy-1,2-dihydrophenyl)acetyl-CoA isomerase [EC:5.3.3.18]
ko:K15849	PAT, AAT	bifunctional aspartate aminotransferase and glutamate/aspartate-prephenate aminotransferase [EC:2.6.1.1 2.6.1.78 2.6.1.79]
ko:K15517	GLYAL	glycine N-phenylacetyltransferase [EC:2.3.1.192]
ko:K14455	GOT2	aspartate aminotransferase, mitochondrial [EC:2.6.1.1]
ko:K14454	GOT1	aspartate aminotransferase, cytoplasmic [EC:2.6.1.1]
ko:K13372	aauB	aralkylamine dehydrogenase heavy chain [EC:1.4.9.2]
ko:K13371	aauA	aralkylamine dehydrogenase light chain [EC:1.4.9.2]
ko:K13064	PTAL	phenylalanine/tyrosine ammonia-lyase [EC:4.3.1.25]
ko:K12732	ARO10	phenylpyruvate decarboxylase [EC:4.1.1.-]
ko:K11358	yhdR	aspartate aminotransferase [EC:2.6.1.1]
ko:K10797	enr	2-enoate reductase [EC:1.3.1.31]
ko:K10775	PAL	phenylalanine ammonia-lyase [EC:4.3.1.24]
ko:K10437	PHACA	phenylacetate 2-hydroxylase [EC:1.14.13.-]
ko:K07253	MIF	phenylpyruvate tautomerase [EC:5.3.2.1]
ko:K05821	ARO9	aromatic amino acid aminotransferase II [EC:2.6.1.58 2.6.1.28]
ko:K05714	mhpC	2-hydroxy-6-oxonona-2,4-dienedioate hydrolase [EC:3.7.1.14]
ko:K05713	mhpB	2,3-dihydroxyphenylpropionate 1,2-dioxygenase [EC:1.13.11.16]
ko:K05712	mhpA	3-(3-hydroxy-phenyl)propionate hydroxylase [EC:1.14.13.127]
ko:K05711	hcaB	2,3-dihydroxy-2,3-dihydrophenylpropionate dehydrogenase [EC:1.3.1.87]
ko:K05710	hcaC	3-phenylpropionate/trans-cinnamate dioxygenase ferredoxin component
ko:K05709	hcaF, hcaA2	3-phenylpropionate/trans-cinnamate dioxygenase subunit beta [EC:1.14.12.19]
ko:K05708	hcaE, hcaA1	3-phenylpropionate/trans-cinnamate dioxygenase subunit alpha [EC:1.14.12.19]
ko:K04073	mhpF	acetaldehyde dehydrogenase [EC:1.2.1.10]
ko:K03782	katG	catalase-peroxidase [EC:1.11.1.21]
ko:K03334	IL4I1	L-amino-acid oxidase [EC:1.4.3.2]
ko:K02618	paaZ	oxepin-CoA hydrolase / 3-oxo-5,6-dehydrosuberyl-CoA semialdehyde dehydrogenase [EC:3.3.2.12 1.2.1.91]
ko:K02615	paaJ	3-oxo-5,6-didehydrosuberyl-CoA/3-oxoadipyl-CoA thiolase [EC:2.3.1.223 2.3.1.174]
ko:K02614	paaI	acyl-CoA thioesterase [EC:3.1.2.-]
ko:K02613	paaE	ring-1,2-phenylacetyl-CoA epoxidase subunit PaaE
ko:K02612	paaD	ring-1,2-phenylacetyl-CoA epoxidase subunit PaaD
ko:K02611	paaC	ring-1,2-phenylacetyl-CoA epoxidase subunit PaaC [EC:1.14.13.149]
ko:K02610	paaB	ring-1,2-phenylacetyl-CoA epoxidase subunit PaaB
ko:K02609	paaA	ring-1,2-phenylacetyl-CoA epoxidase subunit PaaA [EC:1.14.13.149]
ko:K02554	mhpD	2-keto-4-pentenoate hydratase [EC:4.2.1.80]
ko:K01912	paaK	phenylacetate-CoA ligase [EC:6.2.1.30]
ko:K01904	4CL	4-coumarate–CoA ligase [EC:6.2.1.12]
ko:K01692	paaF, echA	enoyl-CoA hydratase [EC:4.2.1.17]
ko:K01666	mhpE	4-hydroxy 2-oxovalerate aldolase [EC:4.1.3.39]
ko:K01593	DDC	aromatic-L-amino-acid decarboxylase [EC:4.1.1.28]
ko:K01451	hipO	hippurate hydrolase [EC:3.5.1.32]
ko:K01426	E3.5.1.4, amiE	amidase [EC:3.5.1.4]
ko:K00838	ARO8	aromatic amino acid aminotransferase I [EC:2.6.1.57 2.6.1.27 2.6.1.5]
ko:K00832	tyrB	aromatic-amino-acid transaminase [EC:2.6.1.57]
ko:K00824	dat	D-alanine transaminase [EC:2.6.1.21]
ko:K00817	hisC	histidinol-phosphate aminotransferase [EC:2.6.1.9]
ko:K00815	TAT	tyrosine aminotransferase [EC:2.6.1.5]
ko:K00813	aspC	aspartate aminotransferase [EC:2.6.1.1]
ko:K00812	aspB	aspartate aminotransferase [EC:2.6.1.1]
ko:K00811	ASP5	aspartate aminotransferase, chloroplastic [EC:2.6.1.1]
ko:K00628	GLYAT	glycine N-acyltransferase / glycine N-benzoyltransferase [EC:2.3.1.13 2.3.1.71]
ko:K00588	E2.1.1.104	caffeoyl-CoA O-methyltransferase [EC:2.1.1.104]
ko:K00529	hcaD	3-phenylpropionate/trans-cinnamate dioxygenase ferredoxin reductase component [EC:1.18.1.3]
ko:K00500	phhA, PAH	phenylalanine-4-hydroxylase [EC:1.14.16.1]
ko:K00487	CYP73A	trans-cinnamate 4-monooxygenase [EC:1.14.13.11]
ko:K00457	HPD, hppD	4-hydroxyphenylpyruvate dioxygenase [EC:1.13.11.27]
ko:K00285	dadA	D-amino-acid dehydrogenase [EC:1.4.5.1]
ko:K00276	AOC3, AOC2, tynA	primary-amine oxidase [EC:1.4.3.21]
ko:K00274	MAO, aofH	monoamine oxidase [EC:1.4.3.4]
ko:K00270	pdh	phenylalanine dehydrogenase [EC:1.4.1.20]
ko:K00146	feaB	phenylacetaldehyde dehydrogenase [EC:1.2.1.39]
ko:K00129	E1.2.1.5	aldehyde dehydrogenase (NAD(P)+) [EC:1.2.1.5]
ko:K00074	paaH, hbd, fadB, mmgB	3-hydroxybutyryl-CoA dehydrogenase [EC:1.1.1.157]
ko:K00055	E1.1.1.90	aryl-alcohol dehydrogenase [EC:1.1.1.90]

Metabolic graph of Phenylalain Metabolism

Next, lets investigate KOs belonging to Phenylalanin metabolism

metabolic_neighbourhood = grepgraph(assoc_kos)
# nitrogenMetabolism.simplified = contractMetab(nitrogenMetab)

metabolic_neighbourhood %>% contractMetab %>% prettifyGraph %>% plot()

p = metabolic_neighbourhood %>%
    contractMetab %>%
    prettifyGraph %>%
    ig2ggplot(., dfOnly=FALSE)

ggplotly(p)

Ideally, I should have the following: * contigs associated with these KOs * their abundance (gDNA) * their expression (mRNA)

But that’ll require me to run the whole pAss procedure for all KOs in this pathway like I did for nitrogen metabolism. Possible for future work

Canonical Pathways associated Phenylalain Metabolism

Lets get all the pathways associated with paaA using the graph DB

assoc_pathways = assoc_kos %>% map_df(ko2path) %>%
    select(p.pathway, p.pathwayname) %>%
    unique

num_assoc_pathways = assoc_pathways %<>%
    rowwise %>%
    mutate(count = path2ko(p.pathway) %>% nrow) %>%
    arrange(desc(count))

What if we look around the neighbouring KOs, we see they are associated with 54 pathways.

assoc_kos_extended = assoc_pathways %>%
    filter(count < 100) %>%
    pull(p.pathway) %>%
    map_df(path2ko) %>%
    pull(KO) %>%
    unique

# assoc_kos_extended %>% length

extended_metabolic_graph = grepgraph(assoc_kos_extended)

num_kos = names(V(extended_metabolic_graph)) %>% grepl("^ko", .) %>% sum
num_cpds = vcount(extended_metabolic_graph) - num_kos
# V(extended_metabolic_graph)

If we include all associated metabolic pathways we will end up with a metabolic network consisting of 3163 nodes with 1409 KOs and 1754 compounds.

Conclusion

That’s too much KOs to look at

paaA in `meta`-pathway

Properties about paaA

If we look at the immediate metabolic neighbourhood surrounding paaA in the meta-pathway built from abundant KOs,

It is associated with two compounds:

Phenylacetyl-CoA
2-(1,2-Epoxy-1,2-dihydrophenyl)acetyl-CoA.

See below:

surrounding = grepgraph(paaA)
graph_topkos = grepgraph(top500kos)
# V(surrounding) %in% V(graph_topkos)

rbind(
      names(V(surrounding)) %>% grep("cpd", ., value=T) %>% map_df(cpdname) %>% as_tibble %>% rename(id=cpd) %>% select(-mass, -weight),
      names(V(surrounding)) %>% grep("ko", ., value=T) %>% map_df(koname) %>% as_tibble %>% rename(id=ko.ko, name=ko.name) %>% select(id, name)
) %>% kable

id	name
cpd:C00582	Phenylacetyl-CoA;
cpd:C20062	2-(1,2-Epoxy-1,2-dihydrophenyl)acetyl-CoA
ko:K02609	paaA

paaA IS a highly abundant KO
paaA is identified as a specialist KO but dominated by 1 genus, Thermus

Now lets highlight paaA (red), in the metabolic network formed by top KOs, paaA is only connected to one other KO, phenylacetate−CoA, ligase and not connected to the main connected component of the AS metabolic network.

graph_topkos = grepgraph(top500kos) %>% contractMetab %>% prettifyGraph() #%>% plot()
graph_topkos_original = graph_topkos
vid = which(V(graph_topkos)$name %>% do.call(c,.) %in% 'ko:K02609')
# V(graph_topkos)$color[vid] =
V(graph_topkos)$color = "grey"
V(graph_topkos)$color[vid] = "red"
V(graph_topkos)$size[vid] = 10
V(graph_topkos)$name = ""
V(graph_topkos)$label = ""
graph_topkos %>% plot

If we were search around the metabolic neighbourhood of paaA, for KOs connected 2 steps away from paaA, (ignoring compounds), as shown in the figure below, we find numerous paths like the one shown below:

Figure: Third degree connected KOs

Red nodes are KOs and grey nodes are compounds

# path = (k1:ko {ko:"ko:K02609"})-->(c1:cpd)-->(k2:ko)-->(c2:cpd)-->(k3:ko{ko:"ko:K01692"})

query = '
MATCH
    path = (k1:ko {ko:"ko:K02609"})-->(c1:cpd)-->(k2:ko)-->(c2:cpd)-->(k3:ko)
WHERE
    id(k1) <> id(k2)
    AND id(k1) <> id(k3)
RETURN
    k2.ko as k2,
    k3.ko as k3
'
outer = dbquery(query)
outer %<>% unique

# outer$k3[outer$k3 %in% names(V(graph_topkos_original))] %>% as.character %>% unique

Neighbouring KOs

#neighbouring kos
neigh = unique(c(as.character(outer$k2), as.character(outer$k3)))
neigh %>% map_df(koname) %>% as_tibble %>% kable

ko.ko	ko.name	ko.definition
ko:K15866	paaG	2-(1,2-epoxy-1,2-dihydrophenyl)acetyl-CoA isomerase [EC:5.3.3.18]
ko:K02613	paaE	ring-1,2-phenylacetyl-CoA epoxidase subunit PaaE
ko:K02612	paaD	ring-1,2-phenylacetyl-CoA epoxidase subunit PaaD
ko:K02611	paaC	ring-1,2-phenylacetyl-CoA epoxidase subunit PaaC [EC:1.14.13.149]
ko:K02610	paaB	ring-1,2-phenylacetyl-CoA epoxidase subunit PaaB
ko:K18363	padD	phenylacetyl-CoA:acceptor oxidoreductase 26-kDa subunit
ko:K18362	padC	phenylacetyl-CoA:acceptor oxidoreductase 27-kDa subunit
ko:K18361	padB	phenylacetyl-CoA:acceptor oxidoreductase [EC:1.17.5.1 3.1.2.25]
ko:K18360	padA	phenylacetyl-CoA:acceptor oxidoreductase accessory protein
ko:K02614	paaI	acyl-CoA thioesterase [EC:3.1.2.-]
ko:K01912	paaK	phenylacetate-CoA ligase [EC:6.2.1.30]
ko:K00628	GLYAT	glycine N-acyltransferase / glycine N-benzoyltransferase [EC:2.3.1.13 2.3.1.71]
ko:K02618	paaZ	oxepin-CoA hydrolase / 3-oxo-5,6-dehydrosuberyl-CoA semialdehyde dehydrogenase [EC:3.3.2.12 1.2.1.91]
ko:K02615	paaJ	3-oxo-5,6-didehydrosuberyl-CoA/3-oxoadipyl-CoA thiolase [EC:2.3.1.223 2.3.1.174]
ko:K07515	HADHA	enoyl-CoA hydratase / long-chain 3-hydroxyacyl-CoA dehydrogenase [EC:4.2.1.17 1.1.1.211]
ko:K07514	EHHADH	enoyl-CoA hydratase / 3-hydroxyacyl-CoA dehydrogenase / 3,2-trans-enoyl-CoA isomerase [EC:4.2.1.17 1.1.1.35 5.3.3.8]
ko:K07511	ECHS1	enoyl-CoA hydratase [EC:4.2.1.17]
ko:K06446	DCAA	acyl-CoA dehydrogenase [EC:1.3.99.-]
ko:K01825	fadB	3-hydroxyacyl-CoA dehydrogenase / enoyl-CoA hydratase / 3-hydroxybutyryl-CoA epimerase / enoyl-CoA isomerase [EC:1.1.1.35 4.2.1.17 5.1.2.3 5.3.3.8]
ko:K01782	fadJ	3-hydroxyacyl-CoA dehydrogenase / enoyl-CoA hydratase / 3-hydroxybutyryl-CoA epimerase [EC:1.1.1.35 4.2.1.17 5.1.2.3]
ko:K01692	paaF, echA	enoyl-CoA hydratase [EC:4.2.1.17]
ko:K15517	GLYAL	glycine N-phenylacetyltransferase [EC:2.3.1.192]
ko:K18359	padH	phenylglyoxylate dehydrogenase epsilon subunit [EC:1.2.1.58]
ko:K18358	padF	phenylglyoxylate dehydrogenase delta subunit [EC:1.2.1.58]
ko:K18357	padE	phenylglyoxylate dehydrogenase gamma subunit [EC:1.2.1.58]
ko:K18356	padI	phenylglyoxylate dehydrogenase beta subunit [EC:1.2.1.58]
ko:K18355	padG	phenylglyoxylate dehydrogenase alpha subunit [EC:1.2.1.58]
ko:K15054	mdlB	(S)-mandelate dehydrogenase [EC:1.1.99.31]
ko:K01576	mdlC	benzoylformate decarboxylase [EC:4.1.1.7]
ko:K10437	PHACA	phenylacetate 2-hydroxylase [EC:1.14.13.-]
ko:K01501	E3.5.5.1	nitrilase [EC:3.5.5.1]
ko:K01426	E3.5.1.4, amiE	amidase [EC:3.5.1.4]
ko:K00146	feaB	phenylacetaldehyde dehydrogenase [EC:1.2.1.39]
ko:K00129	E1.2.1.5	aldehyde dehydrogenase (NAD(P)+) [EC:1.2.1.5]
ko:K01451	hipO	hippurate hydrolase [EC:3.5.1.32]

Let’s overlay these neighbouring KOs with those found in the meta-pathway formed by the top genomically abundant KOs.

paaa_buddies = c('ko:K02609', neigh)
around = paaa_buddies[paaa_buddies %in% names(V(graph_topkos_original))] %>% as.character %>% koname
koi = around$ko.ko %>% as.character
around %>% kable

ko.ko	ko.name	ko.definition
ko:K02609	paaA	ring-1,2-phenylacetyl-CoA epoxidase subunit PaaA [EC:1.14.13.149]
ko:K01912	paaK	phenylacetate-CoA ligase [EC:6.2.1.30]
ko:K01692	paaF, echA	enoyl-CoA hydratase [EC:4.2.1.17]

graph_topkos = graph_topkos_original
vid = which(V(graph_topkos)$name %>% do.call(c,.) %in% koi)
hmmm = which(V(graph_topkos)$label %in% "5-Carboxy-2-pentenoyl-CoA;")

V(graph_topkos)$color = "grey"
V(graph_topkos)$color[vid] = "red"
V(graph_topkos)$color[hmmm] = "green"
V(graph_topkos)$size[vid] = 10
V(graph_topkos)$name = ""
V(graph_topkos)$label = ""
graph_topkos %>% plot

paaA (red) and paaK (red) another subunit, can be connected to enoyl-CoA hydratase, which is in the largest connected component via 5-Carboxy-2-pentenoyl-CoA; through

One connection through 2-(1,2-epoxy-1,2-dihydrophenyl)acetyl-CoA, which is not included in the abundant meta-pathway

query='
MATCH
    path = (k1:ko {ko:"ko:K02609"})-->(c1:cpd)-->(k2:ko { ko:"ko:K15866"})-->(c2:cpd)-->(k3:ko {ko:"ko:K01692"})
return
    k1.ko, c1.cpd, k2.ko, c2.cpd, k3.ko'
dbquery(query) %>% as_tibble %>% kable

k1.ko	c1.cpd	k2.ko	c2.cpd	k3.ko
ko:K02609	cpd:C20062	ko:K15866	cpd:C14144	ko:K01692

graph_topkos = grepgraph(c(top500kos, 'K15866')) %>% contractMetab %>% prettifyGraph() #%>% plot()
vid = which(V(graph_topkos)$name %>% do.call(c,.) %in% c(koi, 'ko:K15866'))
hmmm = which(V(graph_topkos)$label %in% "5-Carboxy-2-pentenoyl-CoA;")
# V(graph_topkos)$color[vid] =
V(graph_topkos)$color = "grey"
V(graph_topkos)$color[vid] = "red"
V(graph_topkos)$color[hmmm] = "green"
V(graph_topkos)$size[vid] = 10
V(graph_topkos)$name = ""
V(graph_topkos)$label = ""
graph_topkos %>% plot

Future Works

We should investigate the gene centric assembly for paaG and see if it is also dominated by contigs labelled as belonging to Thermus.

sessionInfo()

## R version 3.5.1 (2018-07-02)
## Platform: x86_64-conda_cos6-linux-gnu (64-bit)
## Running under: Ubuntu 16.04.5 LTS
## 
## Matrix products: default
## BLAS/LAPACK: /home/jovyan/anaconda3/envs/meta3/lib/R/lib/libRblas.so
## 
## locale:
##  [1] LC_CTYPE=C.UTF-8       LC_NUMERIC=C           LC_TIME=C.UTF-8       
##  [4] LC_COLLATE=C.UTF-8     LC_MONETARY=C.UTF-8    LC_MESSAGES=C.UTF-8   
##  [7] LC_PAPER=C.UTF-8       LC_NAME=C              LC_ADDRESS=C          
## [10] LC_TELEPHONE=C         LC_MEASUREMENT=C.UTF-8 LC_IDENTIFICATION=C   
## 
## attached base packages:
## [1] stats     graphics  grDevices utils     datasets  methods   base     
## 
## other attached packages:
##  [1] plotly_4.8.0     ggplot2_3.1.0    markdown_0.9     knitr_1.21      
##  [5] rmarkdown_1.11   magrittr_1.5     igraph_1.2.4     bindrcpp_0.2.2  
##  [9] purrr_0.2.5      dplyr_0.7.6      MetamapsDB_0.0.2
## 
## loaded via a namespace (and not attached):
##  [1] Biobase_2.42.0              httr_1.3.1                 
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## [77] bindr_0.1.1                 stringi_1.2.4              
## [79] parallel_3.5.1              Rcpp_0.12.18               
## [81] tidyselect_0.2.4            xfun_0.5

# rmarkdown::render("./paaa-analysis.Rmd")

Investigating ring-1,2-phenylacetyl-CoA epoxidase subunit PaaA’s role in Activated Sludge