Purine catabolic pathway in Bacillus subtilis is consisted of more than 14 genes. Among these genes, pucL and pucM are required for uricase activity. While PucL is known to encode the uricase itself, the function of PucM is still unclear although this protein is also indispensable for uric acid decomposition. Here, we provide evidence that PucM, a transthyretin-related protein, functions to facilitate the hydrolysis of 5-hydroxyisourate, the end product of the uricase reaction. Based on these results, we propose that transthyretin-related proteins present in diverse organisms are not functionally related to transthyretin but actually function as a hydroxyisourate hydrolase.

The ureide pathway, which produces ureides from uric acid, is an essential purine catabolic process for storing and transporting the nitrogen fixed in leguminous plants and some bacteria. PucM from Bacillus subtilis was recently characterized and found to catalyze the second reaction of the pathway, hydrolyzing 5-hydroxyisourate (HIU), a product of uricase in the first step. PucM has 121 amino acid residues and shows high sequence similarity to the functionally unrelated protein transthyretin (TTR), a thyroid hormone-binding protein. Therefore, PucM belongs to the TTRrelated proteins (TRP) family. The crystal structures of PucM at 2.0 Å and its complexes with the substrate analogs 8-azaxanthine and 5,6-diaminouracil reveal that even with their overall structure similarity, homotetrameric PucM and TTR are completely different, both in their electrostatic potential and in the size of the active sites located at the dimeric interface. Nevertheless, the absolutely conserved residues across the TRP family, including His-14, Arg-49, His-105, and the C-terminal Tyr-118 -Arg-119 -Gly-120 -Ser-121, indeed form the active site of PucM. Based on the results of sitedirected mutagenesis of these residues, we propose a possible mechanism for HIU hydrolysis. The PucM structure determined for the TRP family leads to the conclusion that diverse members of the TRP family would function similarly to PucM as HIU hydrolase.5-hydroxyisourate hydrolase ͉ Bacillus subtilis ͉ purine catabolism T he purine de novo biosynthesis is the universal, central metabolic process in all organisms. The pathway begins with glutamine and phosphoribosylpyrophosphate and proceeds through multiple sequential enzymatic steps to the end product inosine monophosphate, which is subsequently used as the precursor for the biosynthesis of other purine nucleotides (1, 2). Some bacteria use the nitrogen in purine bases as an energy source under nitrogen-limited conditions. Therefore, the catabolic pathway degrading purine nucleotides has been proposed to be an essential metabolic process (reviewed in refs. 3 and 4). Uric acid, a major intermediate of purine catabolism, can be excreted or subjected to further degradation, depending on the presence of unique enzyme systems in different organisms. Sequential enzymatic reactions using uric acid as a substrate result in ureides, including allantoin and allantoate (Scheme 1) (1,3,4). This ureide pathway plays a vital role in transporting and storing the nitrogen fixed in leguminous plants in the form of ureides, which have a relatively high N-to-C ratio of 1.0. Moreover, symbiotic N 2 -fixing bacteria actively supply the available nitrogen, which is in turn assimilated into glutamine, a substrate in the first step of purine biosynthesis. In the ureide pathway, the conversion of uric acid into allantoin was initially thought to involve a single step catalyzed by urate oxidase (E.C. 1.7.3.3; uricase), but recent investigations have revealed that this pathway includes two additional, distinct, chemically labile intermedi...

In a search for proteins involved in cancer metastasis, we analyzed proteomes of the human gastric cancer cell OCUM-2M and its metastatic subline OCUM-2MLN. We observed that aspartate aminotransferase (AAT), D-site binding protein (DBP), and anterior gradient protein 2 (AGR2) are differentially expressed in metastatic OCUM-2MLN cells. Measurement of protein expression in clinical samples indicated that DBP and AAT are also down-regulated in metastatic adenocarcinoma. Additionally, urokinase-type tissue plasminogen activator is up-regulated in OCUM-2MLN cells and also in metastatic gastric cancer samples. Collectively, these results raise a possibility that AAT, DBP and AGR2 are functionally implicated in the invasiveness of gastric cancer cells.

In the present study, we have investigated the proteome changes associated with glutamate-induced HT22 cell death, a model system to study oxidative stress-mediated toxicity. Among a number of HT22 proteins exhibiting altered expression, several molecular chaperones demonstrated substantial changes. For example, the levels of Hsp90 and Hsp70 decreased as cell death progressed whereas that of Hsp60 increased dramatically. Interestingly, cytosolic Hsp60 increased more prominently than mitochondrial Hsp60. Concomitantly, the accumulation of poly-ubiquitylated proteins and differential regulation of the peptidase activities and the subunits of 26S proteasomes were observed in glutamate-treated HT22 cells. Our findings that the molecular chaperones and the ubiquitin-proteasome system undergo changes during glutamate-induced HT22 cell death may suggest the importance of a protein quality control system in oxidative damage-mediated toxicity.

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