Structural and functional analysis of PucM, a hydrolase in the ureide pathway and a member of the transthyretin-related protein family

Abstract

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...

Proteome Analysis ofArabidopsisLeaf Peroxisomes Reveals Novel Targeting Peptides, Metabolic Pathways, and Defense Mechanisms

“…Except for the tryptic dipeptide T18 (LR) that escaped detection due to its small size, the entire PTS2 nonapeptide was covered by the sequencing analysis, as can be deduced from the fragment ion mass spectrum of T19 (B). By contrast, both shorter splice variants of TLP (TLP 311 and TLP 286 ) lack the internal PTS2 and TLP 286 additionally the bulky conserved His residue (His 209 of TLP 324 , corresponding to His 14 of TLP/ PucM of Bacillus subtilis) that is implicated in determining the entrance diameter of the internal channel (Jung et al, 2006). Thus, the shorter transcriptional variants probably differ in subcellular localization and function from the peroxisomal isoform.…”

“…Uricase, in fact, produces 5-hydroxyisourate (5-HIU) rather than the presumed pathway end product, allantoin. The enzyme that succeeds uricase in purine catabolism and opens the ring (i.e., 5-HIU hydrolase [COG2351]) turned out to be encoded by tlp ( Figure 3A; Lee et al, 2005;Jung et al, 2006). The reaction product is converted to (S)-allantoin by a decarboxylase (COG3195; Ramazzina et al, 2006) that is homologous to the additional N-terminal domain of bifunctional Arabidopsis TLP (Figure 3).…”

Purine Utilization byKlebsiella oxytocaM5al: Genes for Ring-Oxidizing and -Opening Enzymes

“…Sequence comparisons indicate that the HpxT and HpxQ proteins are HIU hydrolase and OHCU decarboxylase, respectively (15,44,77). The HpxT sequence (108 residues; COG2351) is 32% identical to that of HIU hydrolase (PucM protein; 121 residues) from Bacillus subtilis 168 (44,84). The HpxQ sequence (166 residues; COG3195) is 27% identical to that of the amino-terminal 165 residue OHCU decarboxylase domains of urate oxidase (uricase) from Bacillus sp.…”

Deficiency of 5-hydroxyisourate hydrolase causes hepatomegaly and hepatocellular carcinoma in mice

“…S1C). Although not part of the predicted active site at the interface between the subunits of the HIU hydrolase tetramer (13)(14)(15), replacement of the aromatic tyrosine residue with a sulfur-containing cysteine in the Urah Plt2 protein is likely to disrupt this highly ordered hydrophobic core and lead to protein instability.…”

“…S1B), suggesting that this residue may be important for normal protein function. Crystal structures of HIU hydrolase from prokaryotes as well as zebra fish (13)(14)(15) predict that the tyrosine substituted in Urah plt2/plt2 mice (Y98) normally exists in a highly structured helical region and forms a strong set of hydrophobic interactions between the helix and adjacent β-sheet strands (Fig. S1C).…”