The difficulty of annotating genes: the case of putrescine carbamoyltransferase

Daniil G. Naumoff1,{dagger}, Ying Xu2, Victor Stalon2, Nicolas Glansdorff2 and Bernard Labedan1

1 Institut de Génétique et Microbiologie, CNRS UMR 8621, Université Paris Sud, Bâtiment 409, 91405 Orsay Cedex, France
2 Laboratoire de Microbiologie, Université Libre de Bruxelles and Institut de Recherches Microbiologiques J.M. Wiame, 1, ave E. Gryzon, B-1070 Brussels, Belgium

Correspondence
Bernard Labedan
(bernard.labedan{at}igmors.u-psud.fr)


{dagger}Present address: State Institute for Genetics and Selection of Industrial Micro-organisms, I-Dorozhny proezd, 1, Moscow 117545, Russia

Incorrect annotation of genes and enzymes is an important source of confusion in phylogenetic studies and analysis of metabolic networks.

We have recently identified as putrescine carbamoyltransferases (PTCases; EC 2.1.3.6, GO: 0050231) a set of enzymes that have been erroneously annotated as ornithine carbamoyltransferases (EC 2.1.3.3, GO: 0009348) on the basis of genomic data (Naumoff et al., 2004). These PTCases are involved in the catabolism of the polyamine agmatine via the agmatine deiminase system (Simon & Stalon, 1982) present in several Gram-positive bacteria. Accordingly, we have been able to re-annotate the function of the corresponding encoding genes in those bacteria (Enterococcus faecalis, Listeria monocytogenes, Streptococcus mutans and Lactococcus lactis) for which the genomes have been entirely sequenced (Naumoff et al., 2004). This is summarized in Fig. 1 (see upper part, Cluster A) which also displays more recent data confirming and extending our re-annotation process to other bacteria including one distantly related proteobacterium, Photobacterium profundum [re-annotation of the hypothetical ornithine carbamoyltransferase PBPRB1921 (TrEMBL Q6LG11) as a PTCase].



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Fig. 1. Contradictory use of the name aguB. The re-annotation we have proposed (Naumoff et al., 2004) for the set of genes encoding the proteins involved in the agmatine deiminase pathway in four Gram-positive bacteria (Enterococcus faecalis, GenBank/EMBL/DDBJ accession no. AE016830; Listeria monocytogenes, AL591824; Streptococcus mutans, AE014133; Lactococcus lactis, AE005176) has been summarized here and extended to another, more distant, Gram-positive bacterium, Mycoplasma mycoides (GenBank/EMBL/DDBJ accession no. BX293980). The re-annotation proposed by Griswold et al. (2004a) is also indicated in the case of S. mutans. This set of data is framed to indicate the homogeneity of these respective sets of genes (Cluster A). Below this frame the homologues of those genes that we have detected in the newly sequenced genome of the {gamma}-proteobacterium Photobacterium profundum are indicated using the same motif for the respective homologous genes. Finally, we show the cluster of genes present in Pseudomonas aeruginosa (GenBank/EMBL/DDBJ accession no. AE004091) that is involved in agmatine utilization. This unrelated Cluster B contains the actual aguB gene (see text). Note also the homology between the Pseudomonas aeruginosa aguR gene and a previously unannotated Photobacterium profundum gene (PBPRB1919).

 
While our article (Naumoff et al., 2004) was being reviewed, Griswold et al. (2004a) identified independently the SMU.262 gene in S. mutans as encoding a PTCase and re-designated it with the name aguB (Fig. 1). We would like to draw attention to the fact that this renaming is inappropriate and misleading since aguB has been already used by Haas et al. (1984) and more recently by Nakada et al. (2001) and Nakada & Itoh (2003) to designate the gene encoding the N-carbamoylputrescine amidohydrolase (N-carbamoylputrescine amidase, EC 3.5.1.53, GO: 0050126), an enzyme involved in agmatine utilization by Pseudomonas aeruginosa (Mercenier et al., 1980) as summarized in Fig. 1 (see lower part, Cluster B). As a matter of fact, the two enzymes are neither homologous nor analogous. Indeed, PTCase converts carbamoylputrescine to putrescine and carbamoylphosphate, whereas N-carbamoylputrescine amidohydrolase converts N-carbamoylputrescine to putrescine, CO2 and NH3, and their respective amino acid sequences are completely unrelated. Moreover, the cognate genes belong to different genetic contexts (Naumoff et al., 2004, and Fig. 1).

Furthermore, the name aguB has also been used by Voget et al. (2003) to designate a gene encoding a putative {beta}-agarase (see Table 1) identified from a soil metagenome. This adds to the confusion and should be considered an inappropriate designation also.


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Table 1. Summary of the matches found in TrEMBL when querying the UniProt knowledgebase with the text search ‘aguB’

There were no matches in SwissProt.

 
Consequently, we propose the name ptcA to designate the gene encoding PTCase.

It should be stressed that the uncontrolled misuse of the name aguB has created an intolerable chaos in databases, as shown by the following excerpts from the latest version of TrEMBL (release 27.4, 31 August 2004), which is part of the UniProt knowledgebase (Apweiler et al., 2004). This confusion is clearly apparent in Table 1, which was obtained by querying UniProt with the text search ‘aguB’.

In order to prevent people obtaining such misleading and worthless results where three unrelated enzymic activities are recovered when querying with a unique gene name descriptor, we strongly suggest to use the name aguB to designate exclusively genes encoding N-carbamoylputrescine amidohydrolase as we have specified in the column ‘Annotation is:’ of Table 1 since both gene aguB and protein AguB have been well studied for a long time and unambiguously characterized as playing a major role in putrescine biosynthesis as well as in agmatine catabolism (Nakada & Itoh, 2003, and references therein).

With regard to aguA, the use of this name by Griswold et al. (2004a) to designate agmatine deiminase respects the nomenclature proposed by Haas et al. (1984) and more recently by Nakada et al. (2001) and Nakada & Itoh (2003) and is thus correct. By contrast, using aguC for carbamate kinase instead of arcC is again not appropriate since the name arcC is universally recognized as designating carbamate kinases. It must be noted that, in a subsequent article studying in detail the arginine deiminase operon in another Streptococcus species, the same authors went back to using arcC in the case of the corresponding carbamate kinase (Griswold et al., 2004b).

Interestingly, Fig. 1 further shows that a gene homologous to the transcriptional regulator aguR which has been characterized by Nakada et al. (2001) and Nakada & Itoh (2003) as negatively regulating the expression of the aguBA operon in Pseudomonas aeruginosa (cluster B in Fig. 1) is also found in Photobacterium profundum. However, in Photobacterium profundum, this aguR-like gene is inserted in a set of genes that appear to be homologous to the agmatine deiminase gene cluster (cluster A in Fig. 1) found in Gram-positive bacteria and encoding PTCase, amino acid permease, agmatine deiminase and carbamate kinase, respectively (Griswold et al., 2004a; Naumoff et al., 2004), but with a different gene order. This aguR-like gene does not appear to be evolutionarily related to the putative regulator SMU.261c of the agmatine deiminase pathway in S. mutans (Griswold et al., 2004a) nor to its homologues EF0731 (E. faecalis) and llrH (Lactococcus lactis). This would appear to be an interesting instance of recruitment of a regulatory gene from one pathway to another and deserves further study.

Acknowledgements
This work was supported by the CNRS (UMR 8621) and by the Flanders Foundation for Joint and Fundamental Research. D. G. N. was supported by a postdoctoral grant from the French Ministère de la Recherche.

REFERENCES

Apweiler, R., Bairoch, A., Wu, C. H. & 12 other authors (2004). UniProt: the Universal Protein knowledgebase. Nucleic Acids Res 32 Database issue, D115–D119.[CrossRef][Medline]

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Naumoff, D. G., Xu, Y., Glansdorff, N. & Labedan, B. (2004). Retrieving sequences of enzymes experimentally characterized but erroneously annotated: the case of the putrescine carbamoyltransferase. BMC Genomics 5, 52.[CrossRef][Medline]

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Voget, S., Leggewie, C., Uesbeck, A., Raasch, C., Jaeger, K. E. & Streit, W. R. (2003). Prospecting for novel biocatalysts in a soil metagenome. Appl Environ Microbiol 69, 6235–6242.[Abstract/Free Full Text]





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