7584 Nucleic Acids Research, 2007, Vol. 35, No. 22 http://ecocyc.org/enzymes.shtml, which is updated for every EcoCyc release. Finally, we face the problem of dead-end metabolites within the E. coli metabolic network. A dead-end metabolite is a metabolite that is produced by the metabolic network and has no reactions consuming it, or that is only consumed by the metabolic network and has no reactions generating it, and in both cases has no identiﬁed transporter. We authored a program that searches EcoCyc according to a simpliﬁed version of the preceding deﬁnition (see Methods section) and found 169 dead-end metabolites. We focused our analysis on those 33 dead ends that are substrates in metabolic pathways, with the following results. See also analyses of E. coli dead ends by Reed et al. (20) (many of the results from that analysis were previously incorporated into EcoCyc) and by Kumar et al. (21) (which considered a broader set of dead ends, some of which are not found in pathways). Analysis of dead-end compounds within a Pathway/ Genome Database (PGDB) is a useful exercise to identify missing information in the database or in the experimental literature. In the case of EcoCyc, the classiﬁcation of carbamate and H2CO3 as dead-end compounds led to the realization that the EcoCyc cyanate degradation pathway was incorrect. Rather than being depicted as an intermediate, carbamate was shown as a side product of the two-step pathway. The corrected pathway shows carbamate as an unstable intermediate that decomposes in a spontaneous reaction to ammonia and CO2. The ﬁnal step, hydration of CO2 to bicarbonate by carbonic anhydrase, was shown to be an essential component of the pathway to prevent depletion of the intracellular bicarbonate pool (22). A small number of compounds were classiﬁed as dead- end metabolites for trivial reasons. Several compounds are able to diﬀuse freely across the cytoplasmic membrane or are utilized in the periplasm, and thus there is no requirement for pathways or reactions to produce or import them (acetoacetate, dimethyl sulfoxide and dimethylsulﬁde are examples). Other compounds are known to participate in spontaneous reactions; this information allowed us to remove certain compounds (OH-, H2CO3, carbamate) from the list of dead ends. One compound, tetrahydropteroyltri-L-glutamate, lacked the proper chemical classiﬁcation that would have allowed it to be included as a substrate in folate polyglutamylation. The classiﬁcation of a small number of compounds as dead-end metabolites occurred because information that is available in the experimental literature was missing within EcoCyc. Adding appropriate reactions and pathways that produce or utilize these compounds (50-methylthioadeno- sine, undecaprenyl N-acetyl-glucosaminyl-N-acetyl- mannosaminuronate-4-acetamido-4,6-dideoxy-D-galac- tose pyrophosphate, 1,6-anhydro-N-acetylmuramate and GDP-L-fucose) to EcoCyc will be a priority in our further curation eﬀorts. Other cases of dead-end metabolites point to the need for further research. A number of metabolites require transport reactions to make them available to the cytoplasmic enzymes that utilize or degrade them (1,6-anhydro-N-acetylmuramate, 4-methyl-5-(beta- hydroxyethyl)thiazole, cobinamide, ethylene glycol, hydroxymethylpyrimidine, phenylethylamine, psicosely- sine). For example, it is known that E. coli can grow on phenylethylamine, for which no transporter has been identiﬁed. Other compounds, such as cobinamide, are known to be required for biosynthesis of a metabolite (vitamin B12, in this case), but again no transporter has yet been identiﬁed. 1-Amino-propan-2-ol was thought to be its precursor in vitamin B12 biosynthesis, but this may be questionable. The entire biosynthesis route for vitamin B12 requires additional experimental work. In some cases, it is known that a compound is a precursor for the synthesis of an essential metabolite, but no metabolic route has yet been identiﬁed. One such example is pimeloyl-CoA, which is required for biotin biosynthesis in E. coli. Because E. coli does not require pimeloyl-CoA for growth, it must be able to synthesize the compound, but the synthesis route and any enzymes that may be used are currently unknown. S-adenosyl- 4-methylthio-2-oxobutanoate is a side product in biotin biosynthesis, but despite extensive searches of the avail- able literature, we were unable to ﬁnd information on the ultimate fate of this compound. The identities of the sulfur donors in the biotin synthase and lipoate synthase reactions are thought to be iron–sulfur clusters, but some uncertainty remains, and thus the donors were represented as stand-alone compounds in EcoCyc. For several compounds (aminoacetaldehyde, dimethyl- benzimidazole, oxalurate and urate), there is some suggestion or indirect evidence for synthesis or further metabolism, but no experimental conﬁrmation in E. coli is available. The transporters of E. coli Two hundred and thirty-eight distinct transport reactions (transporter activities) are deﬁned in EcoCyc. Those transport activities are assigned to 214 diﬀerent trans- porters, which are encoded by 355 E. coli genes. A total of 180 additional E. coli genes are estimated to code for transporters of unknown function. The 180 substrates and classes of substrates for which E. coli transporters are known to exist are shown in Table 5. Visualizing EcoCyc genome information The Pathway Tools genome browser is one of several tools for visualizing EcoCyc genome information. It can be entered from any EcoCyc gene page (click on the Map Position line) to produce a genome view centered on that gene. It can also be entered from the BioCyc query page (see http://BioCyc.org/server.html) by selecting a chromosome in the Genome Browser line and clicking Submit. Figure 6 shows a view generated from the EcoCyc genome browser. The view shows a region of the genome that is wrapped across several lines; the right side of one line is continued at the left side of the line below it. The navigation panel in the upper left region allows the user to change the magniﬁcation of the diagram (vertical arrows) or to move the viewed region left or