Logical Identification of an Allantoinase Analog (puuE) Recruited from Polysaccharide Deacetylases

Abstract

The hydrolytic cleavage of the hydantoin ring of allantoin, catalyzed by allantoinase, is required for the utilization of the nitrogen present in purine-derived compounds. The allantoinase gene (DAL1), however, is missing in many completely sequenced organisms able to use allantoin as a nitrogen source. Here we show that an alternative allantoinase gene (puuE) can be precisely identified by analyzing its logic relationship with three other genes of the pathway. The novel allantoinase is annotated in structure and sequence data bases as polysaccharide deacetylase for its homology with enzymes that catalyze hydrolytic reactions on chitin or peptidoglycan substrates. The recombinant PuuE protein from Pseudomonas fluorescens exhibits metal-independent allantoinase activity and stereospecificity for the S enantiomer of allantoin. The crystal structures of the protein and of protein-inhibitor complexes reveal an overall similarity with the polysaccharide deacetylase ␤/␣ barrel and remarkable differences in oligomeric assembly and active site geometry. The conserved Asp-His-His metalbinding triad is replaced by Glu-His-Trp, a configuration that is distinctive of PuuE proteins within the protein family. An extra domain at the top of the barrel offers a scaffold for protein tetramerization and forms a small substrate-binding cleft by hiding the large binding groove of polysaccharide deacetylases. Substrate positioning at the active site suggests an acid/base mechanism of catalysis in which only one member of the catalytic pair of polysaccharide deacetylases has been conserved. These data provide a structural rationale for the shifting of substrate specificity that occurred during evolution.

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

“…1). This conjecture was confirmed by Ramazzina et al, who recently identified the HpxB homolog from Pseudomonas fluorescens DSM 50090 as an (S)-allantoin-specific allantoinase (76). They denote this enzyme PuuE.…”

“…employ HpxA allantoin racemase, related to hydantoin racemases (72,94), as well as the HpxB (PuuE) allantoinase, related to polysaccharide deacetylases and described recently by Ramazzina and coworkers (76). Our work in progress indicates further that other previously undescribed enzymes are involved in allantoate catabolism (Fig.…”

“…The HpxB allantoinase sequence is most similar (about 70% identity) to protein sequences typically annotated as "polysaccharide deacetylase" (76), and the HpxA sequence is most similar (up to 62% identity) to protein sequences typically annotated as "hydantoin racemase." Representative examples are shown in Table 4.…”

Transcriptional Regulation of the Gene Cluster Encoding Allantoinase and Guanine Deaminase in Klebsiella pneumoniae

“…The protein encoded by hpxB displays allantoinase activity. The protein encoded by gene hpxB (KPN_ 01787) in K. pneumoniae displayed 89% identity with HpxB of K. oxytoca (28) and 58% identity with the metal-independent allantoinase encoded by puuE in Pseudomonas fluorescens (29). In K. oxytoca, the ortholog hpxB gene was proposed to encode allantoinase since hpxB insertion mutants failed to grow with allantoin as the sole nitrogen source but displayed normal growth with allantoate (28).…”

“…Instead, this protein displays high similarity to PuuE, a novel allantoinase of Pseudomonas fluorescens that is annotated in structure and sequence databases as polysaccharide deacetylase on the basis of its similarity to enzymes that remove Nlinked or O-linked acetyl groups from cell wall polysaccharides (29). As for HpxB, other enzymes of the purine catabolic pathway encoded in K. pneumoniae, such as xanthine oxidase (hpxDE) and uricase (hpxO), do not display similarity to other reported enzymes known to catalyze these reactions (10).…”