As enzymes evolve over time, proteins of similar structure acquire small sequence changes and acquire new activities. What are the key changes in an enzyme’s structure or mechanism that allow this to happen? Researchers from the Hedstrom lab, led by former postdoc Gregory Patton, in collaboration with researchers from the Karolinska Institute, investigated this question in the case of two proteins, inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate reductase (GMPR). The enzymes share similar structural features but carry out different reactions in a cell. Since the two enzymes are in opposing pathways, there could be severe consequences if the enzymes slip and carry out the ‘other’ reaction.

The results, published last month in Nature Cell Biology, argue strongly that the difference is based on the ability of the enzyme to switch between two conformations. A single crystal structure of human GMPR type 2 with IMP and NADPH fortuitously captures three different states, each of which mimics a distinct step in the catalytic cycle of GMPR, including states in which the cofactor (NAD or NADP) is either in an ‘in’ conformation poised for hydride transfer (below, right), or an ‘out’ conformation in which the cofactor is 6 Å from IMP (below, left).

Using mutagenesis along with kinetic experiments, the group demonstrates that the ‘out’ conformation is required for the deamination of GMP. The accessibility of this conformation at the key step in GMPR but not IMPDH seems to determine the two different outcomes — thus, the freedom of the enzyme and cofactor to carry out a conformational change determines the specificity.

An interesting question, looking at the pathways, is whether GMPR can ‘run in reverse’, catalyzing the direct amination of IMP to form GMP (and saving the cell some energy in the process).  Overexpression of GMPR does allow E. coli to survive in the absence of IMPDH and GMPS, demonstrating that GMPR-driven synthesis of GMP can support life.  Indeed, some modern organisms that live in ammonia-rich environments appear to obtain GMP by this strategy.  If life began in an ammonia rich environment as is often proposed, the ancestral purine biosynthetic pathways may have produced GMP via GMPR.

For more details, see the paper:  Patton GC, Stenmark P, Gollapalli DR, Sevastik R, Kursula P, Flodin S, Schuler H, Swales CT, Eklund H, Himo F, Nordlund P, Hedstrom L. Cofactor mobility determines reaction outcome in the IMPDH and GMPR (beta-alpha)(8) barrel enzymes. Nat Chem Biol. 2011.