1. The Biomedical Relevance of Microbial Catabolic Diversity John Archer Department of Genetics University of Cambridge j.archer@gen.cam.ac.uk

2. Free Radical Theory of Aging Harman, 1956 Auto-oxidative damage ultimately impairs metabolic efficiency Prediction: promotion of oxidative reactions will correlate with reduced longevity Genetic factors may promote oxidative stress

3. Metabolism Cells+nutrient+O 2 -> more cells+CO 2 +H 2 O Energy metabolism: derive high energy compounds from carbon-energy source Anabolism: complexity of carbon-containing compounds increases Catabolism: complexity of carbon-containing compounds decreases Enzyme-catalysed catabolism is highly sensitive to oxidative modification of substrate because modified substrates may not bind their cognate enzyme

4. Degenerative Molecular Markers: Characteristics Marker often formed by reactive oxygen species Marker concentration should increase with age Rate of accumulation of the marker should be inversely related to longevity of the organism Genetic factors influence rate of accumulation Aberrant accumulation of marker associated with pathology

5. Degenerative Molecular Markers: Candidates LipofuscinCeroid-lipofuscin Modified lipids (especially cholesterol) in foam cells leading to atherosclerosis N-retinyl-N-retinylidene ethanolamine (A2E) in retinal pigment epithelial cells

6. Degenerative Markers or Causative Agent? Lipofuscin may not be direct cause of aging. At moderate levels it has no effect on RER in neurons, but in high levels (75% of pericarion) is deleterious to neuronal adaptability. LSD are strongly linked to ceroid lipofuscin accumulation. Atheroma is correlated with coronary disease and is a clear causative agent. N-retinyl-N-retinylidene ethanolamine (A2E) in retinal pigment epithelial cells may have a role age-related macular degeneration

7. Enzyme Addition Therapy Degenerative marker compounds accumulate because they are not substrates for normal lysosomal enzymes Degenerative markers do not accumulate in the environment – there must be enzymes which can process these molecules Can one identify enzymes from other living systems that can recognise degenerative marker compounds? Brady et al., mannose-terminal glucocerebrosidase treatment for Gaucher's Disease

8. The Substrate Lipofuscin 30-70% protein (standard amino acids) 20-50% lipid (triglycerides, fatty acids, cholesterol, phospholipids, dolichol, phosphorylated dolichol) Fe, and other heavy metals Autofluorescent compounds 1,4-dihydropyridines, 2- hydroxy-1,2-dihydropyrrol-3-ones? Resistant to lysosomal enzymes

9. Rhodococcus Metabolic Diversity Rhodococcus harmless, Gram-positive Actinomycete mycolic acid bacterium Genome is sequenced >7 Mb Thousands of catabolic genes, specific for a vast range of carbon-energy sources Aliphatic, halogenated hydrocarbons, halogenated aromatics (pentachlorophenol), BTEX, PAH, Nitroaromatics, Lignin-related, alkoxy aromatics, terephthalates, heteroaromatics, steroids, dioxane, tetrahydrofuran etc. etc..

10. Isolation Protocol Rhodococcus is an oligotrophic bacterium, highly adapted to catabolise complex, recalcitrant mixtures of substrates simultaneously (no catabolic repression) Provide 80-100 microMolar lipofuscin as sole carbon- energy source to Rhodococcus strains. Incubate and score.

11. Rhodococcus Catabolism of Lipofuscin Demonstrated Rhodococcus could utilise lipofuscin, or components of lipofuscin, as a carbon-energy source Rhodococcus is a fungal-like bacterium, possesses membrane bound vesicles in which substrates are degraded by membrane associated enzyme complexes It is very probable that the entire spectrum of lipofuscin can be metabolised by Rhodococcus We propose that Rhodococcus can act as a source of xeno- enzymes to augment human metabolism

12. Atheroma Macrophages enter artery wall to recycle modified lipoproteins entrapped Recalcitrant modified lipoprotein products accumulate in foam cell lysosome Lysosomal function impaired Additional macrophage are recruited Aberrant proliferative response by vascular smooth muscle cells Formation of atherosclerotic plaque

13. Rhodococcus and Atherosclerosis Rhodococcus can utilise cholesterol as a sole carbon-energy source Both extracellular and intracellular membrane bound cholesterol oxidases are characterised Reaction catalysed by cholesterol oxidase:- Cholesterol ---> 4-cholesten-3-one We propose that Rhodococcus can act as a source of xeno- enzymes to augment catabolism of atherosclerotic plaque

14. Supporting Indications Cross-talk Problems Substrate specificity of the bacterial xeno-enzyme will restrict the level of cross-talk between the bacterial enzyme and the human metabolism Delivery to lysosomal compartment Mannose-terminal glucocerebrosidase treatment of Gaucher's Disease Lysosomal targeting by glycosylation Acid pH of lysosomal compartment Enzyme properties can be engineered in vitro Immune response Small sample data, but promising so far

15. Steps to Biomedical Application of Xenohydrolases Isolate competent enzymes using a genomics approach Engineer the recombinant protein for lysosomal targeting Partner Competence assay in cell system Murine tests Assay competence in disease models

16. Conclusions Lipofuscin, a degenerative molecular marker and component of several lysosomal storage diseases can be catabolised completely or partially by enzyme(s) encoded by the bacterium Rhodococcus Rhodococcus can catabolise several components of atheroma It is highly likely that recalcitrant lysosomal components can be removed by xeno-enzyme treatment