A gold standard set of mechanistically diverse enzyme superfamilies

Superfamily and family analyses provide an effective tool for the functional classification of proteins, but must be automated for use on large datasets. We describe a 'gold standard' set of enzyme superfamilies, clustered according to specific sequence, structure, and functional criteria, for use in the validation of family and superfamily clustering methods. The gold standard set represents four fold classes and differing clustering difficulties, and includes five superfamilies, 91 families, 4,887 sequences and 282 structures

Caffeine Junkie: an Unprecedented Glutathione S-Transferase- Dependent Oxygenase Required for Caffeine Degradation by Pseudomonas putida CBB5 Downloaded from

c Caffeine and other N-methylated xanthines are natural products found in many foods, beverages, and pharmaceuticals. Therefore, it is not surprising that bacteria have evolved to live on caffeine as a sole carbon and nitrogen source. The caffeine degradation pathway of Pseudomonas putida CBB5 utilizes an unprecedented glutathione-S-transferase-dependent Rieske oxygenase for demethylation of 7-methylxanthine to xanthine, the final step in caffeine N-demethylation. The gene coding this function is unusual, in that the iron-sulfur and non-heme iron domains that compose the normally functional Rieske oxygenase (RO) are encoded by separate proteins. The non-heme iron domain is located in the monooxygenase, ndmC, while the Rieske [2Fe-2S] domain is fused to the RO reductase gene, ndmD. This fusion, however, does not interfere with the interaction of the reductase with N 1 -and N 3 -demethylase RO oxygenases, which are involved in the initial reactions of caffeine degradation. We demonstrate that the N 7 -demethylation reaction absolutely requires a unique, tightly bound protein complex composed of NdmC, NdmD, and NdmE, a novel glutathione-S-transferase (GST). NdmE is proposed to function as a noncatalytic subunit that serves a structural role in the complexation of the oxygenase (NdmC) and Rieske domains (NdmD). Genome analyses found this gene organization of a split RO and GST gene cluster to occur more broadly, implying a larger function for RO-GST protein partners. C affeine (1,3,7-trimethylxanthine) and other N-methylated xanthines are well known for applications in food and as pharmaceuticals that improve lung function for asthmatics and chronic obstructive pulmonary disease (COPD) sufferers. More recently, these compounds have been investigated for use as natural insecticides and in treatments for cancer, septic shock, and functional neutrophil disorders (1-3). Enzymatic methods for producing and degrading these N-methylated xanthines could have broader applications for health through both biosynthesis and environmental remediation of waste and by-products. Therefore, bacteria that have evolved to live on caffeine as the sole carbon and nitrogen source are of interest, as are their metabolic pathways toward N-methylated xanthines. Pseudomonas putida CBB5 degrades caffeine, theophylline (1,3-dimethylxanthine), and related methylxanthines via sequential N-demethylation to xanthine (4-6). The ordered N-demethylation of caffeine to xanthine occurs in three steps catalyzed by enzymes belonging to the Rieske oxygenase (RO) family (5, 6), which are encoded by the Alx operon. Initially, two Rieske, nonheme Fe(II) monooxygenases, NdmA and NdmB, remove the N 1 -and N 3 -methyl groups, respectively, from caffeine to form 7-methylxanthine. Both enzymes require an unusually large 65-kDa redox-dense RO reductase, NdmD, which transfers electrons from NADH to NdmA and NdmB for oxygen activation. The final step in the caffeine degradation pathway is N 7 -demethylation of 7-methylxanthine to xanthine. This N 7 -demethylation activity was inseparable from NdmD after four chromatographic steps (6). A highly enriched protein fraction containing this activity was comprised of NdmD and two additional major protein bands, as visualized by SDS-PAGE. These two additional peptides are encoded by two genes in the Alx operon, labeled orf7 and orf8, which flank ndmD on the CBB5 genome Here, we report that ndmE encodes a new type of GST that is absolutely required for N 7 -demethylation of 7-methylxanthine, the final step of caffeine degradation in P. putida CBB5. The N 7 -demethylase RO is unusual in itself because the iron-sulfur and non-heme iron domains that compose the normally functional oxygenase are encoded by two separate genes. The non-heme iron is contained in NdmC, while the iron-sulfur domain is fused to NdmD. NdmE is proposed to facilitate the formation of the NdmCDE complex, which catalyzes the N 7 -demethylation. This is the first report of a new class of GST-dependent ROs. Additional identification of similar uncharacterized gene clusters within genome databases suggests that there is a more generalized role for GSTs in oxygenation and/or biodegradation

Target selection and annotation for the structural genomics of the amidohydrolase and enolase superfamilies

To study the substrate specificity of enzymes, we use the amidohydrolase and enolase superfamilies as model systems; members of these superfamilies share a common TIM barrel fold and catalyze a wide range of chemical reactions. Here, we describe a collaboration between the Enzyme Specificity Consortium (ENSPEC) and the New York SGX Research Center for Structural Genomics (NYSGXRC) that aims to maximize the structural coverage of the amidohydrolase and enolase superfamilies. Using sequence- and structure-based protein comparisons, we first selected 535 target proteins from a variety of genomes for high-throughput structure determination by X-ray crystallography; 63 of these targets were not previously annotated as superfamily members. To date, 20 unique amidohydrolase and 41 unique enolase structures have been determined, increasing the fraction of sequences in the two superfamilies that can be modeled based on at least 30% sequence identity from 45% to 73%. We present case studies of proteins related to uronate isomerase (an amidohydrolase superfamily member) and mandelate racemase (an enolase superfamily member), to illustrate how this structure-focused approach can be used to generate hypotheses about sequence–structure–function relationships

A gold standard set of mechanistically diverse enzyme superfamilies.

Superfamily and family analyses provide an effective tool for the functional classification of proteins, but must be automated for use on large datasets. We describe a 'gold standard' set of enzyme superfamilies, clustered according to specific sequence, structure, and functional criteria, for use in the validation of family and superfamily clustering methods. The gold standard set represents four fold classes and differing clustering difficulties, and includes five superfamilies, 91 families, 4,887 sequences and 282 structures

Thermostable Cyanuric Acid Hydrolase from Moorella thermoacetica ATCC 39073▿

Cyanuric acid, a metabolic intermediate in the degradation of many s-triazine compounds, is further metabolized by cyanuric acid hydrolase. Cyanuric acid also accumulates in swimming pools due to the breakdown of the sanitizing agents di- and trichloroisocyanuric acid. Structurally stable cyanuric acid hydrolases are being considered for usage in pool water remediation. In this study, cyanuric acid hydrolase from the thermophile Moorella thermoacetica ATCC 39073 was cloned, expressed in Escherichia coli, and purified to homogeneity. The recombinant enzyme was found to have a broader temperature range and greater stability, at both elevated and low temperatures, than previously described cyanuric acid hydrolases. The enzyme had a narrow substrate specificity, acting only on cyanuric acid and N-methylisocyanuric acid. The M. thermoacetica enzyme did not require metals or other discernible cofactors for activity. Cyanuric acid hydrolase from M. thermoacetica is the most promising enzyme to use for cyanuric acid remediation applications