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Cofactor mobility determines reaction outcome in the IMPDH and GMPR (?-?)8 barrel enzymes.

ABSTRACT: Inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate reductase (GMPR) belong to the same structural family, share a common set of catalytic residues and bind the same ligands. The structural and mechanistic features that determine reaction outcome in the IMPDH and GMPR family have not been identified. Here we show that the GMPR reaction uses the same intermediate E-XMP* as IMPDH, but in this reaction the intermediate reacts with ammonia instead of water. 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. The cofactor is found in two conformations: an 'in' conformation poised for hydride transfer and an 'out' conformation in which the cofactor is 6 Å from IMP. Mutagenesis along with substrate and cofactor analog experiments demonstrate that the out conformation is required for the deamination of GMP. Remarkably, the cofactor is part of the catalytic machinery that activates ammonia.

SUBMITTER: Patton GC

PROVIDER: S-EPMC4552316 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Cofactor mobility determines reaction outcome in the IMPDH and GMPR (β-α)8 barrel enzymes.

Patton Gregory C GC Stenmark Pål P Gollapalli Deviprasad R DR Sevastik Robin R Kursula Petri P Flodin Susanne S Schuler Herwig H Swales Colin T CT Eklund Hans H Himo Fahmi F Nordlund Pär P Hedstrom Lizbeth L

Nature chemical biology 20111030 12

Inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate reductase (GMPR) belong to the same structural family, share a common set of catalytic residues and bind the same ligands. The structural and mechanistic features that determine reaction outcome in the IMPDH and GMPR family have not been identified. Here we show that the GMPR reaction uses the same intermediate E-XMP* as IMPDH, but in this reaction the intermediate reacts with ammonia instead of water. A single crystal struc ...[more]

PMID: 22037469

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Project description:The (βa)8-barrel enzyme fold is frequently encountered in nature and catalyzes a multitude of different reactions. It is therefore interesting to evaluate the evolutionary potential of this fold by laboratory experiments. Along these lines, we tested the ability of the (βa)8-barrel to acquire new catalytic functions by an approach combining random mutagenesis and selection in vivo. The genes encoding 52 different phosphate-binding (βa)8-barrel proteins were randomized by error-prone PCR and cloned into an expression plasmid. The resulting mixed repertoire was used to transform different auxotrophic Escherichia coli (E. coli) strains, each lacking an enzyme with a phosphate-containing substrate. After plating of the different transformants on minimal medium, growth was observed only for two strains, lacking either the gene for the serine phosphatase SerB or the phosphoserine aminotransferase SerC. The same variants of point-mutated E. coli genes nanE (encoding a putative N-acetlymannosamine-6-phosphate 2-epimerase) and pdxJ (encoding the pyridoxine 5’-phosphate synthase) were responsible for rescuing both DserB and DserC. Remarkably, all selected PdxJ variants have acquired a 10-residue C-terminal extension from the cloning vector, that is essential for the rescue of DserB and DserC. Unexpectedly, the complementing NanE and PdxJ variants did not catalyze the SerB or Serc reactions in vitro. However, RT-qPCR, RNAseq, and transcriptome analysis showed that they rescue the deletions by enlisting the help of endogenous E. coli enzymes HisB and HisC through exclusive up-regulation of histidine operon transcription. While the promiscuous SerB activity of HisB is well-established, our data indicates that HisC is promiscuous for the SerC reaction, as well. The successful rescue of DserB and DserC through point mutations and recruitment of additional amino acids in NanE and PdxJ provides another example for the adaptability of the (βa)8-barrel fold. To our knowledge, this is the first example of (βa)8-barrels functioning not as enzymes, but by activating transcription via an unknown mechanism.

Dataset Information

Cofactor mobility determines reaction outcome in the IMPDH and GMPR (?-?)8 barrel enzymes.

Publications

Cofactor mobility determines reaction outcome in the IMPDH and GMPR (β-α)8 barrel enzymes.

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