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Experimental Technology Development for
Metabolism/Metabolite Discovery
Matthew Vetting, Yuriy Patskovsky, Rahul
Bhosle, Jungwook Kim, Rafael Toro,
Nawar al Obaidi, Steve Almo ([email protected])
Enzyme Function Initiative (EFI)
ASBMB Workshop
April 29, 2014
Infrastructure/automation
STRUCTURES: TOTAL 106; NEW FOR TARGET 79, LIGANDED 66
11/12-
10/13 Clones
Soluble
Constructs
Unique
Soluble
Purification
Attempts Shipments
Total 3127 1775 1050 967 852
Aeromonas hydrophila
Aeromonas hydrophila
Rhizobium radiobacter
Anaeromyxobacter dehalogenans
Archaeoglobus fulgidus
Bacillus anthracis
Bacillus cereus
Bacillus halodurans
Bacillus subtilis
Bacillus thuringiensis
Bacteroides fragilis
Bacteroides thetaiotaomicron
Bacteroides vulgatus
Bartonella henselae
Bordetella bronchiseptica
Borrelia burgdorferi
Bordetella parapertussis
Bordetella pertussis
Burkholderia ambifaria
Campylobacter jejuni
Campylobacter jejuni
Campylobacter jejuni
Chromobacterium violaceum
Clostridium acetobutylicum
Clostridium difficile
Clostridium perfringens
Clostridium thermocellum
Colwellia psychrerythraea
Corynebacterium glutamicum
Cryptosporidium Parvum
Cytophaga hutchinsonii
Deinococcus radiodurans
Desulfovibrio vulgaris
Enterococcus faecalis
Escherichia coli
Escherichia coli
Escherichia coli
Geobacter metallireducens
Giardia intestinalis
Haemophilus ducreyi
Haemophilus influenzae
Haloarcula marismortui
Idiomarina loihiensis
Klebsiella pneumoniae
Lactobacillus acidophilus
Lactobacillus brevis
Lactobacillus plantarum
Lactococcus lactis
Legionella pneumophila
Listeria innocua
Marinobacter aquaeolei
Mesoplasma florum
Methanocaldococcus jannaschii
Methanococcus maripaludis
Methanosarcina mazei
Methylobacillus flagellatus
Methylococcus capsulatus
Methylococcus capsulatus
Mycobacterium tuberculosis
Neisseria gonorrhoeae
Neisseria meningitidis
Novosphingobium aromaticivorans
Oenococcus oeni
Pediococcus pentosaceus
Porphyromonas gingivalis
Pseudomonas aeruginosa
Pseudomonas fluorescens
Pseudomonas putida
Pseudomonas syringae pv.
phaseolicola
Pseudomonas syringae pv. tomato
Pyrococcus horikoshii
Ralstonia solanacearum
Rhodopseudomonas palustris
Rhodospirillum rubrum
Salinibacter ruber
Salmonella enterica
Salmonella typhimurium
Ruegeria pomeroyi
Ruegeria pomeroyi
Staphylococcus saprophyticus
Streptococcus agalactiae
Streptococcus mutans
Streptococcus pneumoniae
Streptococcus thermophilus
Streptomyces avermitilis
Streptomyces violaceoruber
Sulfolobus solfataricus
Thermoplasma acidophilum
Thermoplasma volcanium
Thermus thermophilus
Thermus thermophilus
Vibrio cholerae
Vibrio parahaemolyticus
Xanthomonas campestris
Xylella fastidiosa
Elizabethkingia miricola
Acidothermus cellulolyticus
Actinobacillus succinogenes
Arthrobacter aurescens
Arthrobacter chlorophenolicus
Ashbya gossypii
Bacteroides stercoris
Bacillus licheniformis
Rhizobium radiobacter
Prochlorococcus sp.
Prochlorococcus sp.
Prochlorococcus sp.
Prochlorococcus sp.
Burkholderia cenocepacia
Burkholderia thailandensis
Chloroflexus aurantiacus
Citrobacter freundii
Prochlorococcus sp.
Dyadobacter fermentans
Escherichia coli
Geobacter sulfurreducens
Gloeobacter violaceus
Gluconobacter oxydans
Ralstonia eutropha
Hyphomonas neptunium
Lactobacillus delbrueckii
Leptospira interrogans serovar
Copenhageni
Mycobacterium avium paratuberculosis
Mycobacterium marinum
Mycobacterium smegmatis
Streptomyces cattleya
Nocardioides sp.
Prochlorococcus sp.
Polaromonas sp.
Pseudoalteromonas atlantica
Pseudomonas putida
Rhodococcus opacus
Bradyrhizobium sp.
Renibacterium salmoninarum
Rhizobium etli
Rhodobacter sphaeroides
Rhodobacter sphaeroides
Rhodoferax ferrireducens
Saccharopolyspora erythraea
Salinispora tropica
Shewanella oneidensis
Prochlorococcus sp.
Prochlorococcus sp.
Prochlorococcus sp.
Sulfitobacter sp.
Sulfitobacter sp.
Sulfolobus acidocaldarius
Thermobifida fusca
Thermus thermophilus
Treponema denticola
Verminephrobacter eiseniae
Wolinella succinogenes
Xanthobacter autotrophicus
Streptomyces griseus
Halomicrobium mukohataei
Spirosoma linguale
Haloferax volcanii
Desulfovibrio alaskensis
Zymomonas mobilis
Prochlorococcus sp.
Acinetobacter baumannii
Candida albicans
Lodderomyces elongisporus
Debaryomyces hansenii
Candida glabrata
Scheffersomyces stipitis
Kluyveromyces lactis
Methylobacterium radiotolerans
Saccharomyces cerevisiae
Sinorhizobium meliloti
Sinorhizobium meliloti
Prochlorococcus marinus
Prochlorococcus marinus
Prochlorococcus Myovirus
Prochlorococcus Myovirus
Xenorhabdus bovienii
Xenorhabdus nematophila
Photorhabdus luminescens
Chitinophaga pinensis
Halogeometricum borinquense
Planctomyces limnophilus
Flavobacterium johnsoniae
Desulfovibrio desulfuricans
Methanoculleus marisnigri
Natronomonas pharaonis
Thermomonospora curvata
Micrococcus luteus
Pyrococcus furiosus
Lactobacillus reuteri
Thermotoga lettingae
Sulfurimonas denitrificans
Escherichia fergusonii
Capnocytophaga gingivalis
Meiothermus ruber
Akkermansia muciniphila
Methanococcus vannielii
Methanothermobacter
thermautotrophicus
Methanococcus aeolicus
Porphyromonas gingivalis
Methanocorpusculum labreanum
Fusobacterium nucleatum
Acholeplasma laidlawii
Porphyromonas endodontalis
Prevotella melaninogenica
Flavobacterium psychrophilum
Desulfovibrio salexigens
Acidithiobacillus ferrooxidans
Actinobacillus pleuropneumoniae
Anaerococcus prevotii
Bacillus selenitireducens
Bifidobacterium longum subsp. infantis
Brachybacterium faecium
Burkholderia graminis
Clostridium phytofermentans
Finegoldia magna
Geodermatophilus obscurus
Gluconacetobacter diazotrophicus
Gordonia bronchialis
Jonesia denitrificans
Leptotrichia buccalis
Neisseria meningitidis
Ochrobactrum anthropi
Parabacteroides distasonis
Pedobacter heparinus
Pseudomonas aeruginosa
Psychrobacter cryohalolentis
Sebaldella termitidis
Shewanella loihica
Staphylococcus epidermidis
Streptobacillus moniliformis
Streptococcus mitis
Streptosporangium roseum
Sulfurospirillum deleyianum
Thermanaerovibrio acidaminovorans
Thermotoga maritima
Thermotoga neapolitana
Thermotoga petrophila
Tsukamurella paurometabola
Veillonella parvula
Chromohalobacter salexigens
Methanosarcina acetivorans
Acinetobacter baumannii
Anabaena variabilis
Bacillus cereus
Bacillus thuringiensis serovar
israelensis
Bartonella bacilliformis
Brevibacterium
Burkholderia multivorans
Campylobacter coli
Campylobacter concisus
Campylobacter curvus
Campylobacter lari
Campylobacter upsaliensis
Caulobacter vibrioides
Corynebacterium diphtheriae
Citrobacter koseri
Enterococcus faecium
Francisella philomiragia subsp.
philomiragia
Lactobacillus casei
Listeria monocytogenes
Magnetospirillum magneticum
Mannheimia haemolytica
Methanococcus maripaludis
Methanococcus voltae
Methylibium petroleiphilum
Moorella thermoacetica
Mycobacterium tuberculosis
Nostoc punctiforme
Nostoc sp.
Pectobacterium atrosepticum
Photobacterium profundum
Rhizobium vitis
Rhodopseudomonas palustris
Rhodopseudomonas palustris
Roseobacter denitrificans
Salmonella enterica subsp. arizonae
Salmonella enterica subsp. enterica
Shewanella pealeana
Shewanella putrefaciens
Shigella flexneri
Staphylococcus aureus
Streptococcus agalactiae
Streptococcus gordonii
Streptococcus pneumoniae
Streptococcus pneumoniae
Streptococcus pyogenes
Streptococcus pyogenes
Streptococcus sanguinis
Streptococcus thermophilus
Streptococcus uberis
Synechococcus elongatus
Thermoanaerobacter sp.
Thiobacillus denitrificans
Vibrio fischeri
Kineococcus radiotolerans
Methylobacterium extorquens
Shewanella woodyi
Cupriavidus metallidurans
Shewanella denitrificans
Saccharophagus degradans
Granulibacter bethesdensis
Cellulomonas flavigena
Rhodothermus marinus
Leuconostoc mesenteroides
Heliobacterium modesticaldum
Lactobacillus gasseri
Eggerthella lenta
Methanobrevibacter smithii
Cryptobacterium curtum
Metallosphaera sedula
Lactobacillus delbrueckii
Atopobium rimae
Mycobacterium abscessus
Weissella paramesenteroides
Thermoanaerobacter pseudethanolicus
Genomic DNA from ~340 organisms
YOU can help!
• Nitrogen Fixation by Sinorhizobium (model symbiotic
organism; Michael Kahn, WSU; Allen Orville, BNL;
Sharon Long, Stanford; Mary Lipton, PNNL)
• Carbon fixation by Prochlorococcus (most abundant
organism on Earth; Penny Chisholm, MIT)
• Biofuels via Phaeodactylum tricornutum (JCVI)
• Human gut microbiota (Jeff Gordon, Wash U.)
Prochlorococcus
Sinorhizobium
200 µm
Phaeodactylum
Gut Communities
Relevant to NIH and DOE Missions
Systems biology/environmental science
Collaboration for symbiotic systems biology:
Sinorhizobium meliloti:Alfalfa
• Model organism for Nitrogen Fixation and Symbiosis: Michael Kahn,
WSU; Allen Orville, BNL; Sharon Long, Stanford; Mary Lipton, PNNL)
Sinorhizobium
Alfalfa
root
Bacteroids
200 µm
R. Dixon & D. Kahn Nature Rev. Microbiol. 2:621-631 (2004)
Prokaryote: S. meliloti • Uses RCHx → ATP + e-
• Differentiated for N2 → NH3
• 6218 protein genes
• ORFeom cloned
Eukaryote: M. truncatulata: • Root nodules • Supplies RCHx uses NH3
• Creates microaerobic cond. • Genome recently sequenced • 47,845 genes • Young et al. Nature 480, 520–524 (2011)
Oct 2011: WSU and PNNL (M.L. Kahn & M.S. Lipton) complete mass-tag, metabolomic and microproteomic analysis of N2-fixing root nodules.
• S. meliloti express about 1200 proteins (many already in process) • M. truncatulata expresses about 1500 proteins • About 140 small molecule metabolites identified
Systems biology of symbiosis
Prokaryote ↔ Eukaryote
Bacteroids
200 µm
R. Dixon & D. Kahn Nature Rev. Microbiol. 2:621-631 (2004)
Prokaryote: S. meliloti • Uses RCHx → ATP + e-
• Differentiated for N2 → NH3
• 6218 protein genes
• ORFeom cloned
Eukaryote: M. truncatulata: • Root nodules • Supplies RCHx uses NH3
• Creates microaerobic cond. • Genome recently sequenced • 47,845 genes • Young et al. Nature 480, 520–524 (2011)
Oct 2011: WSU and PNNL (M.L. Kahn & M.S. Lipton) complete mass-tag, metabolomic and microproteomic analysis of N2-fixing root nodules.
• S. meliloti express about 1200 proteins (many already in pipeline) • M. truncatulata expresses about 1500 proteins • About 140 small molecule metabolites identified
TARGETS CLONED EXPRESS PURIFIED
Sino. 4109 2338 1025 449
N2F 2322 1559 639 411
Systems biology of symbiosis
Prokaryote ↔ Eukaryote
Gluconokinase Putative 2-dehydro-3-
deoxygalactonokinase
Probable tagatose 6-
phosphate kinase
Adenosine kinase Glycerol kinase Probable sugar kinase Probable sugar kinase
Sinorhizobium and N2F sugar kinases
Aerobic
Anaerobic structural genomics
B C
D E F
All bacterial expression
Large-scale expression of radical SAMs
Immune regulators
Small-scale BV expression of cytoplasmic Adhesive Proteins
Large-scale BV expression of IgSF Large-scale HEK293
expression of Fc-fusions
Small-scale BV expression validation of secreted proteins
Eukaryotic expression platforms
Phosphorylated substrates (167) are arranged in the screen by “class.”
Deb Mariano (U of NM), Karen Allen (BU)
High-throughput functional screening: HADs
HADSF enzyme from Bacillus halodurans; annotated as a hydrolase, shows broad-range specificity with high activity.
Deb Mariano (U of NM), Karen Allen (BU)
Identified enzymes that are promiscuous
PDB: 4JB3 BT_0970 “HAD-like phosphatase” • Bacteroides thetaiotaomicron • Specific for short carbon chain sugars • Cap closed conformation • Best hit: D-ribitol 5-phosphate
Discovery of a “missing” enzyme from a
well-known pathway
Deb Mariano (U of NM), Karen Allen (BU)
Discovery of a “missing” enzyme from a
well-known pathway
GTP cyclohydrolase II RibA
2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5’-phosphate deaminase
5-amino-6-ribosylamino-2,4(1H,3H)-
pyrimidinedione 5’-phosphate deaminase
unknown phosphatase
RibD
riboflavin kinase RibF
FAD synthase RibF
riboflavin synthase RibC
6,7-dimethyl-8-ribityllumazine
synthase
RibB
RibE
FAD FMN
riboflavin
Deb Mariano (U of NM), Karen Allen (BU)
Discovery of a “missing” enzyme from a
well-known pathway
Mulligan, C et al. (2011). "Tripartite ATP-
independent periplasmic (TRAP)
transporters in bacteria and archaea."
FEMS Microbiol Rev 35(1): 68-86.
1ANF
ABC SBP
Maltose
Binding Protein
3B50
TRAP SBP
Sialic acid
Binding Protein
1ZAY
LacI SBP
Full Length
Purine Repressor
2F78
LysR SBP
Benzoate
Transcriptional
Regulator
SBP Domain
Solute Binding Proteins (SBP) as entries into
Operon Function Discovery
Number of Initial Targets 304
Targets with score of 2-3 (Are Soluble) 203 (66% of Targets)
HTP Assayed Targets 160
Number of Targets with Hit (> 4x deviation of control) 91 (66% of assayed targets)
Number of Targets with Hit > 5 degrees 74 (54% of assayed targets)
Number of Targets with No Hits 47 (34% of assayed targets)
How useful are SBPs?
heat
0
20000
40000
60000
80000
100000
120000
140000
20 25 30 35 40 45 50 55 60
RF
U
temp (0C)
DMSO (ctrl)
JAK3 Inhibitor
VI
Rho Kinase
Inhibitor III,
Rockout
AG 1478
Thermal denaturation monitored by fluorescence
heat
0
20000
40000
60000
80000
100000
120000
140000
20 25 30 35 40 45 50 55 60
RF
U
temp (0C)
DMSO (ctrl)
JAK3 Inhibitor
VI
Rho Kinase
Inhibitor III,
Rockout
AG 1478
Thermal denaturation monitored by fluorescence
Number of Initial Targets 304
Targets with score of 2-3 (Are Soluble) 203 (66% of Targets)
Thermofluor Assayed Targets 160
191 Compounds in 184 Wells + 8 Controls; All in Duplicate
34 L/D Amino Acids (Tyrosine) 4 Long Chain Dicarboxylic Acids (Adipate)
30 L/D Aldoses (Ribose) 5 Short Chain Dicarboxylates (Fumarate)
39 L/D Mono-Acid Sugars (Gluconate) 4 Keto Acids (Pyruvate, Lactate)
17 L/D Di-Acid Sugars (Glucarate) 6 Other Sugars (Fructose, Sucrose, Maltose)
16 L/D Uronic Acids (Glucuronate) 4 Amino Sugars (Glucosamine)
18 Aromatic benzoate derivatives (p-Coumarate) 5 Modified Amino Acids (pyroglutamate)
7 polyols (Ribitol) 2 Dipeptides (D-Ala-D-Ala)
5 Osmolytes (Ectoin, Betaine, Proline Betaine)
How many are soluble, how many have been
screened, what’s in the ThermoFluor Screen?
Cluster 1-9-X bind uronates
Abundance of TRAP SBP Xtals
510075 with bound D-Ala-D-Ala
510075 D-Ala-D-Ala Binding Protein
SBP TRAP PROTEINS SETUP AS APO
“MYSTERY DENSITY”
2.5 sigma omit map
Orotate?
510109 (Desal_2161) Desulfovibrio salexigens
2157 2158 2159 2160 2161 2162 2163 2164 2165 2166
TRAP transporter
Dihydroorotate oxidase
N-carbamoyl-L-aspartate amidohydrolase (an orphan enzyme)
Dihydroorotase Peptidase U32
Transcriptional regulator Orotate
phosphoribosyltransferase
Two possible outcomes
GlpA-C glycerol-3-phosphate dehydrogenase (anaerobic)
2.5 sigma omit map
510107 and 510113 are not related (20% seq ID)
Glycerol 3-Phosphate
510107 (Ddes_1525) Desulfovibrio desulfuricans
RT: 0.00 - 90.02 SM: 13G
0 10 20 30 40 50 60 70 80 90
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relativ
e Abund
ance
NL: 5.79E3
m/z= 173.02013-173.02187 F: FTMS + c ESI Full ms [78.50-1000.00] MS 1mMsnGlycerolPO4
RT: 0.00 - 90.00 SM: 13G
0 10 20 30 40 50 60 70 80
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relativ
e Abun
dance
NL: 1.57E2
m/z= 173.02013-173.02187 F: FTMS + c ESI Full ms [78.50-1000.00] MS 510107A
RT: 0.00 - 90.01 SM: 13G
0 10 20 30 40 50 60 70 80 90
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relativ
e Abun
dance
NL: 1.39E2
m/z= 173.02013-173.02187 F: FTMS + c ESI Full ms [78.50-1000.00] MS 510113
RT: 0.00 - 90.00 SM: 13G
0 10 20 30 40 50 60 70 80 90
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relative
Abunda
nce
NL: 2.38E2
m/z= 173.02013-173.02187 F: FTMS + c ESI Full ms [78.50-1000.00] MS 510120
sn-G3P Std*
510107**
510113
510120**
*sn-glycerol-3-phosphate standard **SBPs 510107 and 510120 have peaks for the mass 172.0137 (sn-G3P?)
sn-G3P
sn-glycerol-3-phosphate (sn-G3P, exact mass:
172.0137)
FMN; unknown function
Methanobacterium
CDP; riboflavin kinase
Methanococcus
Alanine; signal receiver
Vibrio cholera
Glucose; methyltransferase
Streptcoccus
Lipid; unknown function
Geobacillus
OPP; unknown function
E. coli
Fortuitous examples
HTP expression, purification, structural and functional characterization
We want your target lists!
Summary
