Structure of mammalian ornithine decarboxylase at 1.6 Å resolution: stereochemical implications of PLP-dependent amino acid decarboxylases

Self Cite

The Epstein-Barr virus thwarts immune surveillance through a Gly-Ala repeat (GAr) within the viral EpsteinBarr virus-encoded nuclear antigen 1 protein. The GAr inhibits proteasome processing, an early step in antigen peptide presentation, but the mechanism of proteasome inhibition has been unclear. By embedding a GAr within ornithine decarboxylase, a natural proteasome substrate that does not require ubiquitin conjugation, we now demonstrate inhibition in a purified system, excluding involvement of ubiquitin conjugation or of proteins extraneous to substrate and proteasome. We show further that the GAr acts as a stop-transfer signal in proteasome substrate processing, resulting in vivo in partial proteolysis that halts just short of the GAr. Similarly, introducing a GAr into green fluorescent protein destabilized by the ornithine decarboxylase degradation domain also stops the progress of proteolysis, leading to the accumulation of partial degradation products. We postulate that the ATP motor of the proteasome slips when it encounters the GAr, impeding further insertion and, in this way, halting degradation.

Section: Discussionmentioning

confidence: 99%

Repeat Sequence of Epstein-Barr Virus-encoded Nuclear Antigen 1 Protein Interrupts Proteasome Substrate Processing

Zhang

Coffino

2004

Self Cite

“…Amino acids 131 and 145 may be critical contact residues in one or both of these roles, interacting with ODC or the proteasome. The crystal structure of truncated (22) or fulllength ODC containing all 461 amino acids (23) shows little electron density corresponding to amino acids C-terminal to residue 420, implying that the most distal part of ODC is disordered. It remains to be determined whether this region is more or less flexible when ODC is complexed with AZ1.…”

Section: Discussionmentioning

confidence: 99%

Structural Elements of Antizymes 1 and 2 Are Required for Proteasomal Degradation of Ornithine Decarboxylase

Chen

MacDonald

Coffino

2002

Self Cite

The antizymes constitute a conserved gene family with at least three mammalian orthologs. As described previously, in a degradation system utilizing rabbit reticulocyte lysate, antizyme 1 (AZ1) accelerates proteasomal ornithine decarboxylase (ODC) degradation, but antizyme 2 (AZ2) does not. To examine the relationship between antizyme structure and function, we further characterized the properties of AZ1 and AZ2 and protein chimeras composed of elements of the two. AZ1 binds to ODC with about a 3-fold higher potency than AZ2, but this cannot account for their distinct degradative activities. The dissimilar degradative capacity of AZ1 and AZ2 is also observed using purified proteasomes. A series of reciprocal AZ1/ AZ2 chimeras was used to determine the sequence elements needed to direct ODC degradation. An element contained within amino acids 130 -145 of AZ1 is essential for this function. Constructs in which amino acids 130 -145 were exchanged between the antizymes confirmed the critical nature of this region. Within this region, amino acids 131 and 145 proved responsible for the functional difference between the two forms of AZ.

“…These include eukaryotic ODCs, plant ADC, and a number of bacterial enzymes that encompass a wide range of substrate specificities (arginine, ornithine, lysine, diaminopimelate, and carboxynorspermidine decarboxylases) (20). The x-ray structures of several eukaryotic ODCs (mouse, T. brucei, and human) (23)(24)(25)(26)(27) and of bacterial diaminopime-late decarboxylase have been solved (28). The N-terminal domain forms a ␤/␣-barrel, and the C-terminal domain is folded into a ␤-barrel structure.…”

mentioning

confidence: 99%

Paramecium bursaria Chlorella Virus-1 Encodes an Unusual Arginine Decarboxylase That Is a Close Homolog of Eukaryotic Ornithine Decarboxylases

Shah¹,

Coleman

Mir³

et al. 2004

Paramecium bursaria chlorella virus (PBCV-1) is a large double-stranded DNA virus that infects chlorellalike green algae. The virus encodes a homolog of eukaryotic ornithine decarboxylase (ODC) that was previously demonstrated to be capable of decarboxylating L-ornithine. However, the active site of this enzyme contains a key amino acid substitution (Glu for Asp) of a residue that interacts with the ␦-amino group of ornithine analogs in the x-ray structures of ODC. To determine whether this active-site change affects substrate specificity, kinetic analysis of the PBCV-1 decarboxylase (PBCV-1 DC) on three basic amino acids was undertaken. The k cat /K m for L-arginine is 550-fold higher than for either L-ornithine or L-lysine, which were decarboxylated with similar efficiency. In addition, ␣-difluoromethylarginine was a more potent inhibitor of the enzyme than ␣-difluoromethylornithine. Mass spectrometric analysis demonstrated that inactivation was consistent with the formation of a covalent adduct at Cys 347 . These data demonstrate that PBCV-1 DC should be reclassified as an arginine decarboxylase. The eukaryotic ODCs, as well as PBCV-1 DC, are only distantly related to the bacterial and plant arginine decarboxylases from their common ␤/␣-fold class; thus, the finding that PBCV-1 DC prefers L-arginine to L-ornithine was unexpected based on evolutionary analysis. Mutational analysis was carried out to determine whether the Asp-toGlu substitution at position 296 (position 332 in Trypanosoma brucei ODC) conferred the change in substrate specificity. This residue was found to be an important determinant of substrate binding for both Larginine and L-ornithine, but it is not sufficient to encode the change in substrate preference.