Team:Edinburgh/Cellulases (C model)

How does this whole synergy thing even work? It can seem almost magical that having enzymes closer together can increase their usefulness, but consider the following statements about cellulose degradation, all of which are true ([http://www.annualreviews.org/doi/abs/10.1146/annurev-biochem-091208-085603 Fontes and Gilbert, 2010])

1. Exoglucanase chews away at the end of a cellulase chain, producing cellobiose sugars.
2. Endoglucanase cuts cellulose chains in the middle, essentially turning one chain into two.
3. Cellobiose inhibits the action of the above enzymes.
4. β-glucosidase cuts cellobiose in half, producing two glucose molecules.

Facts 1 and 2 mean that exoglucanase works best with endoglucanase nearby, since every time endoglucanase acts, it produces new chain-ends for exoglucanase to attack. Meanwhile, facts 1, 3, and 4 mean that β-glucosidase helps nearby copies of the other enzymes by preventing the cellobiose from inhibiting them.

Syn: a simple demonstration of synergy

We can demonstrate that there is (potentially) a huge difference between the synergistic and non-synergistic systems with Edinburgh's Syn C program. This is a 2D simulation that works in the following way:

• The world is a 2D grid.
  • Each spot in the grid can contain a sugar, a bond between sugars, or nothing.
  • The world begins with an array of cellulose.
• Cellulose is modelled as horizontal chains with alternating sugars and bonds: s-b-s-b-s-b-s-b-s-b-s, etc.
• There are three enzyme types: endoglucanase, exoglucanase, and β-glucosidase.
  • The enzymes move about randomly, in a "brownian motion" manner.
  • If an enzyme is in the same place as a bond, it can destroy it:
    • Endoglucanase can only cut bonds away from the ends of a chain.
    • Exoglucanase can only cut bonds if this results in a cellobiose molecule (s-b-s) forming.
    • β-glucosidase can only cut cellobiose bonds.
• There is inhibition of exoglucanase by cellobiose; it does not cut bonds (or has a reduced chance to do so) if there is a nearby cellobiose molecule.

Sugars are dark green squares, bonds between sugars are light green. From left to right are shown the actions of endoglucanase, exoglucanase, and β-glucosidase.

Simulating non-synergy vs. synergy

We can run two different simulations with the same settings. However, in one simulation the enzymes float about freely, whereas in the other they travel in triplets, each triplet containing one of each type of enzyme, side by side:

The enzymes' current positions are displayed as white, yellow, or blue squares. Note that in the synergistic system (right) they travel together. Bonus points if you can tell which enzyme is which!

So, what happens if we run a large simulation? This:

Non-synergistic versus synergistic systems. Dark regions are places where the cellulose has been degraded down to free glucose molecules.

Results and conclusion

We carried out 100 runs of the program with standard settings (20 of each enzyme per side, width and height 155, full inhibition of exoglucanase by cellobiose, endoglucanase requiring at least 5 sugars on each side of a bond to cut it).

On average, it took the synergistic system 7522 iterations to liberate half the sugar, as opposed to 13878 for the non-synergistic system.

Synergy - it actually makes sense!

C code

• syn.c
• Makefile

There are two ways to compile the code. The first is to just compile the C file as it stands, using a command like "gcc syn.c". When compiled in this way, there is no graphical display, except that the program saves .bmp graphics files every so often.

However, if you have the SDL graphics library development files (called libsdl1.2-dev in Ubuntu), you can optionally download the Makefile and compile with "make". This version does not (by default) spit out any files; instead, it gives a graphical display of the action as it occurs.

Note that, to work, the Makefile should be called "Makefile" and the C file should be called "syn.c".

Command-line options

The following command-line options should work. Default values are shown:

--halfwidth 155 — width of each simulation

--height 155 — height of the simulations

--margin 4 — size of the margin (region of no cellulose)

--enzymes 20 — number of enzymes per simulation

--bioseinhibleft 1.0 — strength of cellobiose inhibition of exoglucanase (left simulation), from 0 to 1

--bioseinhibright 1.0 — strength of cellobiose inhibition of exoglucanase (right simulation), from 0 to 1

--endoreqleft 5 — how close to the end of a chain endoglucanase can cut (left simulation)

--endoreqright 5 — how close to the end of a chain endoglucanase can cut (right simulation)

--bmpmag 1 — magnification of .bmp files

--sdlmag 2 — magnification of SDL window

--reporttick 250 — how often to print count of glucose and cellobiose to the console (0 for never)

--sdltick 1 — how often to draw to the SDL window (1 to draw every frame)

--delay 20 — milliseconds to delay after drawing to SDL window; some machines require this

The default values of the following settings depend on whether it has been compiled in SDL mode or not:

--bmptick x — how often to spit out .bmp graphics files (0 for never)

--endpoint x — how many iterations to run (0 for unlimited)

Finally, the following options require no value:

--nosyn — do not use synergy on either side

--bothsyn — use synergy on both sides

References

• Fontes CMGA, Gilbert HJ (2010) [http://www.annualreviews.org/doi/abs/10.1146/annurev-biochem-091208-085603 Cellulosomes: highly efficient nanomachines designed to deconstruct plant cell wall complex carbohydrates]. Annual Review of Biochemistry 79: 655-81 (doi: 10.1146/annurev-biochem-091208-085603).