The evolution and structural anatomy of small molecule metabolism

							The evolution and structural
 anatomy of small molecule
  metabolism pathways in
      Escherichia coli.
 Of Pathways and Proteins
  Stuart 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

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    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
           Y-BLAST
                                       +              =
                     Y-BLAST (>75aa)




Proteins              Evolutionary         Pathways
                      Relationships
       Domain assignments
              566 SMM proteins
                                    124 unassigned
                                       proteins
            442 proteins assigned
            to 1+ families (78%)



169 PDB-D families        31 ‘sequence’ domain
                                 families

             200 domain families
                   Glycogen Catabolism
    a-amylase, 3.2.1.1 a-amylase, 3.2.1.1         glycogen phosphorylase

          malS               amyA                           glgP


         Domains
      Glycosyltransferases                                     phosphoglucomutase,   5.4.2.2
                             malodextrin phosphorylase
     a-amylase, and
   ChemistryC-term
      Isozymes
Internal duplication
        substrate
   close substrate
     b-glucosyltransferase              malP                                 pgm
      Phosphoglucomutase


                   malodextrin glucosidase     amylomaltase, 2.4.1.25
                             malZ                       malQ
 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 families
510 domains have arisen by duplication

• 232 duplications within pathways to 278
  duplications across pathways

(Assumption: duplication within pathways
  wherever possible.)
                                         Type of conservation
                                               Internal
                                    Cofactor              Isozymes Chemistry   Substrate
                              100
                                                 Dup.
                              90
Number of proteins involved




                              80
                              70
                              60
                              50
                              40
                              30
                              20
                              10
                               0
      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