macie – a database of enzyme reaction mechanisms janet thornton embl-ebi july 2006
TRANSCRIPT
MACiE – a Database of Enzyme Reaction
Mechanisms
Janet Thornton EMBL-EBI July 2006
Enzymes in Data Resources (2005)
No. enzymes Total No. %-tagein DB
UniProt 65,076 172,690 37.7
PDB (all entries) 14,143 31,522 44.9PDB (non-redundant) 3,655 10,450 35.0
Reactome (human) (via UniProt) 230 680 33.8
Roman Laskowski
Relating the number of enzymes to proteome size
Proteome size
Nu
mb
er o
f en
zym
es
Permissive set
KEGG assignmentsConservative set
human
mouse
wormfly
Shiri Freilich
Increase in Number of Different Reactions (E.C.) in larger proteomes
E.C. 1
E.C. 2
E.C. 3
E.C. 4
E.C. 5
E.C. 6Freilich et al (2005)
JMB ??
Extension and Evolution of Pathways:The integration of the steroid biosynthesis pathway into the sterol biosynthesis pathway
sterol
cholesterol
steroid hormonebile acid
Universalmetazoahuman
Shiri Freilich
Enzyme Structure, Function and Evolution
Outstanding Research Questions:– How is catalysis performed
• Principles of catalysis?
• E.C. numbers
– How do enzymes evolve?
– Can we predict enzyme function from structure?
– Can we design new enzymes?
– What is the enzyme complement in different organisms and how does it evolve?
TrypsinNeed a list of active sites
What constitutes a catalytic residue?
• Direct involvement in the reaction mechanism
• Polarises or alters the pKa of a residue or water
molecule which is directly involved in the reaction mechanism
• Polarises or activates part of the substrate (e.g. making a bond more susceptible to
cleavage)
• Stabilisation of a transition-state intermediate
http://www.ebi.ac.uk/thornton-srv/databases/CSA
The Catalytic Site Atlas: a resource of catalytic sites and residues identified in enzymes using structural data.
Porter, Bartlett, & Thornton Nucl. Acids. Res. (2004) 32: D129-D133.
-lactamase Class A; EC 3.5.2.6; PDB: 1btl– Reaction: -lactam + H2O -amino acid
– Active site residues: S70, K73, S130, E16
N
O
OH
N H 2
OH
S e r
L y s
S e r
N H 3 +
O
H
O
N
O
S e r
L y s
S e r
N H 3 +
O
O
NH
O
O
O
OH
H
S e r
L y s
S e r
G l u
OO H
O
OHO
NH
O
H
N H
S e r
L y s
S e r
G l u
Comparison of CSA, SwissProt & PDB (2004)
Porter, Bartlett et al, 2004 NAR
Conformational Change
Templates
Spherical HarmonicsBinding Site Diversity
Metabolome
Ligand Selectivity
Catalytic Site Atlas
CSA Coverage and Annotations Generated
• Current entries in CSA from Literature = 737
• Current proteins in UniProt annotated by homology = 14,863
• Functional annotations by homology are more accurate if catalytic residues are checked and conserved (George et al (2005) PNAS)
BUT no possibility of storing or querying the proposed chemical mechanisms (which must be available to identify the catalytic residues in the CSA)
The MACiE Database - a Research Project
Mechanism, Annotation and Classification in Enzymes
G. L. Holliday, G. J. Bartlett, Daniel AlmonacidP. Murray-Rust, J.M.Thornton J. B. O. Mitchell
(Holliday et al Bioinformatics 2005 21:4315)
http://www.ebi.ac.uk/thornton-srv/databases/MACiE
Why develop MACiE?
• To understand more about catalysis– To gather information on mechanisms
– To compare and contrast mechanisms in different proteins
– To help validate enzyme mechanisms
– To study the evolution of mechanisms
– To develop mechanism-based classification of enzymes
– To help predict mechanism from structure
– To help design new enzymes
http://www.ebi.ac.uk/thornton-srv/databases/MACiE
The MACiE Database
Content in MACiE● Enzyme Name: fructose-bisphosphate aldolase
● E.C. Classification: (EC 4.1.2.13)– Obsolete EC codes associated with entry: EC 4.1.2.7
● Reference Structure: PDB 1b57– Domain classification: CATH 3.20.20.70– UniProt code: P11604– Specie: Escherichia coli (Bacteria)– Cofactors: Zn2+ and Na+– Catalytic residues: Asp109, Glu182, Asn286
● Links
Classifying Residue Catalytic Function
Hydrogen Donor, Hydrogen bond acceptor, Proton Relay
Nucleophile, Electrophile
Radical relay, Hydride relay
Radical Donor, Radical stabiliser
Leaving group , Steric role, Charge stabiliser
Covalently attached, Metal ligand
Overall Reaction fructose-bisphosphate aldolase
O
O
O
O
O
P
O O -
O -
O
PO -
O -
O
H
HH
O
O O P
O
O -
H
O -
O
O P
O
O -O -
H
HO
+
glycerone phosphate D-glyceraldehyde 3-phosphate D-fructose 1,6-bisphosphate+
Step Annotation in MACiEStep 1: Reactants
O O
Asp 1 0 9
H
Z n 2 +
O
N Asn 2 8 6HH
O -
OGlu 1 8 2
O
H
H
O
H
O P
O
O -
O -
Na +
Step 1: Mechanism
O O
Asp 1 0 9
H
Zn 2 +
O
N Asn 2 8 6HH
O -
OGlu 1 8 2
O
H
H
O
H
O P
O
O -
O -
N a+
Me c ha n is m :P roton tra ns fe r
Ke to -e n o l ta u to m e risa t io n (a ssiste d )
O O
HO
N Asn 2 8 6HH
O
OG lu1 8 2
HOH
O -
H
O P
O
O -
O -O H
O
O
H
PO
O -
O -
OH
OO
HPOO -
O -
M ec h an is m C om po ne nts :Ove rall su b str ate us edIn term e d iate Fo rm e dBon d F or m ed = O-HBon d C lea ve d = C- H
Bo n d (s) ch a n ge d in Ord e r = C -C,1 to C =C , 2 C =O ,2 to C-O , 1
Zn 2 +
N a+
Asp 1 0 9
Step 1: Cofactors
O O
Asp 1 0 9
H
Zn 2 +
O
N Asn 2 8 6HH
O -
OGlu 1 8 2
O
H
H
O
H
O P
O
O -
O -
N a+
Me c ha n is m :P roton tra ns fe r
Ke to -e n o l ta u to m e risa t io n (a ssiste d )
O O
HO
N Asn 2 8 6HH
O
OG lu1 8 2
HOH
O -
H
O P
O
O -
O -O H
O
O
H
PO
O -
O -
OH
OO
HPOO -
O -
M ec h an is m C om po ne nts :Ove rall su b str ate us edIn term e d iate Fo rm e dBon d F or m ed = O-HBon d C lea ve d = C- H
Bo n d (s) ch a n ge d in Ord e r = C -C,1 to C =C , 2 C =O ,2 to C-O , 1
C o fa cto r
C o fa cto r
Zn 2 +
N a+
Asp 1 0 9
Step 1: Spectator Residues
O O
Asp 1 0 9
H
Zn 2 +
O
N Asn 2 8 6HH
O -
OGlu 1 8 2
O
H
H
O
H
O P
O
O -
O -
N a+
Sp e cta to rS id e C h ain
H yd ro ge n Bo n d Acce p to r
S pe ctatorS ide C h a in
Hyd ro g en Bon d Acc ep torH yd rog e n Bo n d D on o r
T ran sitio n S ta te S ta b il ise r
Me c ha n is m :P roton tra ns fe r
Ke to -e n o l ta u to m e risa t io n (a ssiste d )
O O
HO
N Asn 2 8 6HH
O
OG lu1 8 2
HOH
O -
H
O P
O
O -
O -O H
O
O
H
PO
O -
O -
OH
OO
HPOO -
O -
M ec h an is m C om po ne nts :Ove rall su b str ate us edIn term e d iate Fo rm e dBon d F or m ed = O-HBon d C lea ve d = C- H
Bo n d (s) ch a n ge d in Ord e r = C -C,1 to C =C , 2 C =O ,2 to C-O , 1
C o fa cto r
C o fa cto r
Zn 2 +
N a+
Asp 1 0 9
Step 1: Reactant Residues
O O
Asp 1 0 9
H
Zn 2 +
O
N Asn 2 8 6HH
O -
OGlu 1 8 2
O
H
H
O
H
O P
O
O -
O -
N a+
R e a cta ntS ide Ch a in
Pro to n Acce p to r
Sp e cta to rS id e C h ain
H yd rog e n Bo n d Acce p to r
S pe ctato rS ide C h a in
Hyd ro g e n Bon d A ccep torH yd ro ge n Bo n d D on o r
T ran sitio n S ta te S ta b ilise r
Me c ha n is m :P roton tra ns fe r
Ke to -e n o l ta u to m e risa t io n (a ssiste d )
O O
HO
N Asn 2 8 6HH
O
OGlu 1 8 2
HOH
O -
H
O P
O
O -
O -O H
O
O
H
PO
O -
O -
OH
OO
HPOO -
O -
M ec h an is m C om po ne nts :Ove ra ll su b st rate u sedIn term e d iate Fo rm e dBon d F o rm ed = O-HBon d C lea ve d = C -H
Bo n d (s) ch a n ge d in Ord e r = C -C ,1 to C=C , 2 C =O ,2 to C -O, 1
C o fa cto r
C o fa cto r
Z n2 +
N a +
Asp 1 0 9
Similarly Step 2is annotated
Where the information is availablethe rate determining step is annotated
O O
Asp 1 0 9
HO
N Asn 2 8 6HH
O
OGlu 1 8 2
HOH
O -
H
O P
O
O -
O -O H
O
O
H
PO
O -
O -
O -O
Asp 1 0 9
O
N Asn 2 8 6H
H
O
OGlu 1 8 2
H
Sp ec ta to rS id e C ha in
Zn 2 + C o fa cto r
N a + C o fa cto r
Sp ecta to rS id e Ch a in
C h ar ge S ta bil iserH yd ro ge n B o n d D o no r
S te ric Ro le
R e a cta n tS ide Ch a in
P roton Do n o rH ydro g e n Bo nd A cce ptor
H yd ro ge n Bo n d D on o r
R a te D et erm iningSte p
Me c ha nis m:B im ole cu la r N u cleo p h ilic Ad d ition
Pro ton T ra nsfe rA ld o l Add itio n
M e ch an is m C om po ne nts :Ove ra ll s ub st rate u se d
Ove ra ll pro d u ct Fo rm e dInte rm e dia te T er m in a te dBon d F orm ed = C -C , O -H
Bo n d C lea ve d = O-HBo nd (s) cha n g ed in Ord er = C =C , 2 to C -C , 1 C-O , 1 to C =O , 2 C= O, 2 to C -O , 1
OH
O
H
O P
O
O -
O -
O
HO
O
H
PO
O -
O -
H
O
H
H
N a + Co factor
Z n 2 + C ofa cto r
Step 3 is annotated
O O
Asp 1 0 9
H
Z n2 +
O
N Asn 2 8 6HH
O -
OGlu 1 8 2
O
H
H
O
H
O P
O
O -
O -
N a +
O - O
As p1 0 9
O
N Asn 2 8 6H
H
O
OGlu 1 8 2
H
O
H
H
O
HH
O H
O
PO
O -O -
M e ch a nism :Proton transfer
N a + Cofactor
Zn 2 + Cofactor
SpectatorSide Chain
ReactantSide Chain
Proton DonorHydrogen Bond Donor
ReactantSide Chain
Proton AcceptorHydrogen Bond Acceptor
M e c h an is m C o m po ne nt s:Proton Relay
Bond Formed = O-HBond Cleaved = O-H
Infe rre d Re tu rnS te p
MACiE always endeavours to return the enzymeto its ground state.
This is often inferred, which is noted in the annotation
Finally: any spontaneous changes are included
OH
O
H
O P
O
O -
O -
O
HO
O
H
PO
O -
O -
H
Occurs outside enzyme OO
O
O
O
PO O -
O -O
PO -
O -
O
H
HH
These are often spontaneous and occur outside the enzyme
There is no other annotation involved in steps like this
Complete Reaction Annotation
O O
Asp1 0 9
H
Zn 2 +
O
N Asn 2 8 6HH
O -
OG lu 1 8 2
O
H
H
O
H
O P
O
O -
O -
N a +
R ea ctan tS id e C h ain
Proto n Acce pto r
Spe ctatorS ide C h a in
Hyd ro g e n Bon d A cce ptor
Sp ec ta to rS id e C ha in
H yd rog e n Bo n d Acce p to rH yd ro g en B on d D o n or
Tra n sit io n S ta te S tab ilise r
M e c ha nis m :Pro to n tra n sfer
Keto -e no l ta u to m e risa tio n (as siste d)
O O
Asp 1 0 9
HO
N A sn2 8 6HH
O
OGlu 1 8 2
HOH
O -
H
O P
O
O -
O -O H
O
O
H
PO
O -
O -
O - O
Asp 1 0 9
O
N Asn 2 8 6H
H
O
OG lu 1 8 2
H
OH
OO
HPOO -
O -
M e c ha nism C o mp one nt s:Ove ra ll su bs tra te u se dIn te rm e dia te F or m edBo n d Fo rm e d = O -HBo n d Cle a ve d = C -H
Bon d (s) c ha n g ed in Ord er = C -C ,1 to C =C , 2 C =O,2 to C -O, 1
Sp e cta to rS id e C h a in
C o factor
Co fa ctor
Z n2 + C ofa cto r
Na + Co fac to r
Sp e cta to rSid e C h ain
Ch a rg e S ta b ilise rHyd ro g e n Bo nd Do n o r
Ste ric R ole
Re a cta n tS id e C ha in
Pro to n D on o rH yd rog e n Bo n d Acce p to r
Hyd ro g en Bon d D o n or
Ra te D e te rm inin gSte p
M e ch a nis m :Bim o le cu lar N ucle o p hil ic Ad ditio n
Pro to n Tra n sfe rA ld o l Ad d it io n
M e c ha nism Co m pon en ts :O vera ll su b stra te use d
O vera ll p ro du ct Fo rm e dIn te rm e d iate Te rm in ate d
Bo n d Fo rm e d = C -C , O-HBo nd Cle a ved = O- H
Bo n d(s ) ch a ng e d in Ord e r = C =C, 2 to C -C, 1 C -O, 1 to C= O, 2 C =O, 2 to C -O, 1
OH
O
H
O P
O
O -
O -
O
HO
O
H
PO
O -
O -
H
O
H
H
O ccu rs o u ts id e e nzym e
O
HH
O H
O
PO
O -O -
Me c ha nis m:Pro to n t ra nsfer
Na + C ofa cto r
Zn 2 + C o fa cto r
Sp e cta to rS id e C h ain
Re a ctan tS id e C h ain
Pro to n D o no rHyd ro g e n Bo nd Do n or
R e a cta n tS id e Ch a in
Pro to n Acce p to rH ydro g e n Bo nd Acce ptor
Me c ha n is m Co m pon e nts :Pro to n R e la y
Bo n d Fo rm e d = O-HBo n d Cle a ve d = O -H
OO
O
O
O
PO O -
O -O
PO -
O -
O
H
HH
Inf erre d R e turnSte p
Searching MACiE
General Searches
● Query MACiE by reaction comments
● Query MACiE by enzyme and species (scientific and common) names
● Query the chemical changes in MACiE
● Overall reactants and products (by KEGG and ChEBI compound id or compound name)
Frequencies of amino acid reactants performing a given function
Combining the amino acid and functional clusterings
No reactantFunction
No strong preference for
function
Strong preference for acid/base
function
Roles of catalytic residues and mechanistic steps in homologous enzymes of
different function
Gail Bartlett
– How do enzymes modify the chemical reaction they catalyse, using the same structural scaffold?
– Do catalytic residues conserve their role and / or identity in enzyme-catalysed reactions?
Methods
178 enzyme dataset with
assigned catalytic residues and
proposed mechanism
PSIBLAST run against NRDB + PDB (cutoff
e=10-5)
Twenty-seven pairs of homologous
proteins of totally different function
(at primary EC level)
Structural alignment performed using SSM server
Structurally equivalent catalytic
residues Information manually extracted from literature
Catalytic mechanisms
Comparison of function, active site, catalytic
residues and catalytic
mechanism
Results - overview• 27 pairs of proteins• 3 enzyme / nonenzyme pairs• 24 enzyme / enzyme pairs, from 21
enzyme superfamilies
Change of function (EC) class
Function YFunction X
EC 1 EC 2 EC 3 EC 4 EC 5 EC 6 Nonenzyme
EC 1 (oxidoreductases)
- 0 3 3 0 1 2
EC 2 (transferases) - 2 1 1 0 0
EC 3 (hydrolases) - 3 1 0 1
EC 4 (lyases) - 7 1 0
EC 5 (isomerases) - 0 1
EC 6 (ligases) - 0
Enzyme / enzyme pairs
• All but one enzyme pair have their active site located at the same place in the protein fold
• Substrates and / or products shared by 11 pairs
• Cofactor shared by 5 enzyme pairs
Metal ion binding sites
Conserved metal ion type
Altered metal ion type
Conserved metal ion function
5 0
Altered metal ion function
3 4
Twelve enzyme pairs conserve metal binding sites and ligands to the metal ions are structurally aligned
Where the metal ion type has altered, subtle mutations to the ligand binding site have occurred
Rubredoxin oxygen oxidoreductase / metallo--
lactamaseMetallo--lactamase uses a Zn2+-activated hydroxyl for nucleophilic attack on the -lactam substrate
Rubredoxin oxygen oxidoreductase
reduces dioxygen via a redox cycle at a di-
iron site Fe ligand Zn2+ ligand
His 79 His 82
Glu 81 His 84
Asp 83 Asp 86
His 146 His 145
Asp 165 Cys 164
His 226 His 206
Catalytic residue pairs
0
5
10
15
20
25
30
35
Identical role Different role Role in one protein only
Role of residue
Nu
mb
er o
f pai
rs
Unconserved residueidentity
Conserved residueidentity
Catalytic residue pairs
Change in residue identity and residue function
Endonuclease IV / Xylose isomerase
DNA hydrolysis Xylose Xylulose
Change in residue identity and residue function
Trp 136His 109
Endonuclease IV Xylose isomerase
Zn2+-assisted hydroxyl performs
nucleophilic attack on DNA
backbone
Base catalysed ring opening, followed by
intramolecular hydride
transfer and ring closure
Evolution of MechanismMechanisms share a common step at the beginning of the overall reaction pathway, catalysed by residues which are structurally equivalent in both enzymes
Mechanisms share a common step somewhere in the middle of the overall reaction path, catalysed by residues which are structurally equivalent in both enzymes
Mechanisms share common steps at the beginning and end of the overall reaction path, catalysed by residues which are structurally equivalent in both enzymes, but have a different step in the middle
Mechanisms do not share any common steps catalysed by structurally equivalent residues
7
7
2
8
Bartlett et al JMB
Common first stepdehydroquinate synthase / glycerol dehydrogenase
O
O
OPO3
OH
O2-C
OHH
HZn
2+OH
H
NAD+
O2-COOH
OH HOH
OH
OH
OH
H Zn2+O
H
H
NAD+
OH
OH
O
a. Dehydroquinate synthase
b. Glycerol dehydrogenase
several stages
Common first stepdehydroquinate synthase / glycerol dehydrogenase
Superposition of Zn2+ and
ligands
dehydroquinate synthase
H271
E194
H287
H275
glycerol dehydrogenaseH255
H252
D169
H269
Conclusions
• Enzymes are economical in their use of active site residues and features
• It is more likely for residues conserving function to also conserve their identity
• Residues not conserving function tend to mutate
• Tend to find common mechanistic steps at the beginning and ‘middle’ of reaction paths – possibly the most energetically difficult step or intermediate is conserved
Future
• Increase coverage in MACiE
• Analyse Catalytic Mechanisms – Ingold Reaction Types; effects of non-polar
residues
• Evolution of enzymes, pathways & metabolism in different organisms & tissues
• Design??
Acknowledgements• Gail Bartlett, Craig Porter, Jonathan Barker
• The MACiE Team – Cambridge Univ Chemistry DeptJohn Mitchell, Daniel Almonacid, Peter Murray-Rust
• BBSRC, MRC, Wellcome Trust, EMBL
James Torrance Alex Gutteridge Gemma Holliday