The evolution and structural anatomy of

small molecule metabolism pathways in

Escherichia coli.Of Pathways and ProteinsStuart Rison and Sarah

Teichmann

Questions

• How are homologous proteins (enzymes) distributed in E. coli metabolism?

• How does this distribution fit with theories of pathway evolution?

Pathway evolution

• Norman Horowitz, 1945: ‘On the evolution of biochemical syntheses’, Proc. Nat. Acc. Sci. 31:153-157.

“Retrograde evolution”• Roy Jensen, 1976: ‘Enzyme

recruitment in evolution of new function’, Ann. Rev. Microbiol 30:409-425.

“Patchwork evolution”

Retrograde evolution

[ ]

[ ]

[ ]

Jensen, 1976: Substrate ambiguity

• ‘Original pool’ of unregulated and enzymatically versatile proteins

• Enzymes recruited from the pool• Ad hoc pathways• Gene duplication and

specialisation leads to regulated, specific and efficient pathways

Patchwork evolution

Why E. coli?

• An extensively studied model organism

• Complete genome available• Most Small Molecule Metabolism

pathways well known and empirically characterised

• A manageable size

• Good associated databases

Strategy

• Identify all SMM proteins and the pathway(s) in which they belong

• Detect homologous proteins by structure or sequence

• Combine these data to analyse homologous protein distribution in SMM

Methods

E. coli

IMPALA

HMM

-BLAST

-BLAST (>75aa)

+ =

EvolutionaryRelationships

PathwaysProteins

566 SMM proteins

442 proteins assignedto 1+ families (78%)

124 unassignedproteins

169 PDB-D families 31 ‘sequence’ domainfamilies

200 domain families

Domain assignments

Chemistry andclose substrateChemistry and

substrate

Glycogen Catabolism

malQ

malS

malZ

pgmmalP

glgPamyA

-amylase, 3.2.1.1

phosphoglucomutase, 5.4.2.2

amylomaltase, 2.4.1.25

-amylase, 3.2.1.1 glycogen phosphorylase

malodextrin phosphorylase

malodextrin glucosidase

-glucosyltransferase

Phosphoglucomutase

-amylase, C-term

Glycosyltransferases

Domains

Internal duplicationIsozymes

Duplications Across Pathways

• 110 out of 200 families occur in more than one pathway

• Can exhibit conservation of chemistry, shared cofactor or minor substrate similarity

• 36 families have close conservation of EC number (Chemistry conserved)

• 74 families conserve 1 or no EC number; 11 are cofactor-binding families (cofactor, minor substrate)

Duplications within and across Pathways

• 710 domains in 200 families510 domains have arisen by

duplication

• 232 duplications within pathways to 278 duplications across pathways

(Assumption: duplication within pathways wherever possible.)

0

10

20

30

40

50

60

70

80

90

100

Num

ber o

f pro

tein

s in

volv

ed

Cofactor ChemistryIsozymesInternal

Dup.Substrate

Type of conservation

Conclusion: Structural Anatomy

• 710 domains in 442 proteins of the 566 proteins in E. coli SMM pathways

• 200 families (3.5 members/family)

• Most sizeable families are distributed in several pathways

Conclusion: Recruitment and Conservation

• Duplications have taken place between and within pathways to roughly the same degree

• Duplications occur within most longer pathways:– Isozymes, internal duplications and co-

factor binding most common– Chemistry common– Conservation of substrate binding with

modified chemistry is rare

Conclusions: Pathway evolution

• Data support a “patchwork evolution” model

• Little evidence of “retrograde evolution”

Conclusions: hum…

• Recruitment, duplication and evolution of enzymes are constantly taking place so we are always observing a dynamic system

• Likely to be other evolutionary mechanisms and combinations thereof

Future

• Identification and analysis of novel pathway duplication events

• Focus on order in pathways:– Stepwise analysis– Doublet/triplet analysis

• Analysis domain combination in SMM

Acknowledgements

• Sarah A. Teichmann, Dept. Biochemistry, University College London

• Janet M. Thornton, David Lee, Dept. Crystallography, Birkbeck College and Dept. Biochemistry, University College London

• Monica Riley, Alida Pelegrini-Toole, Marine Biology Laboratory, Woods Hole, USA

• Cyrus Chothia, Julian Gough, MRC Laboratory of Molecular Biology, Cambridge, UK