ATP-binding Cassette (ABC) Transport System Solute-binding Protein-guided Identification of Novel d-Altritol and Galactitol Catabolic Pathways in Agrobacterium tumefaciens C58.

Innovations in the discovery of the functions of uncharacterized proteins/enzymes have become increasingly important as advances in sequencing technology flood protein databases with an exponentially growing number of open reading frames. This study documents one such innovation developed by the Enzyme Function Initiative (EFI; U54GM093342), the use of solute-binding proteins for transport systems to identify novel metabolic pathways. In a previous study, this strategy was applied to the tripartite ATP-independent periplasmic transporters. Here, we apply this strategy to the ATP-binding cassette transporters and report the discovery of novel catabolic pathways for d-altritol and galactitol in Agrobacterium tumefaciens C58. These efforts resulted in the description of three novel enzymatic reactions as follows: 1) oxidation of d-altritol to d-tagatose via a dehydrogenase in Pfam family PF00107, a previously unknown reaction; 2) phosphorylation of d-tagatose to d-tagatose 6-phosphate via a kinase in Pfam family PF00294, a previously orphan EC number; and 3) epimerization of d-tagatose 6-phosphate C-4 to d-fructose 6-phosphate via a member of Pfam family PF08013, another previously unknown reaction. The epimerization reaction catalyzed by a member of PF08013 is especially noteworthy, because the functions of members of PF08013 have been unknown. These discoveries were assisted by the following two synergistic bioinformatics web tools made available by the Enzyme Function Initiative: the EFI-Enzyme Similarity Tool and the EFI-Genome Neighborhood Tool.

Experimental strategies for functional annotation and metabolism discovery: targeted screening of solute binding proteins and unbiased panning of metabolomes.

The rate at which genome sequencing data is accruing demands enhanced methods for functional annotation and metabolism discovery. Solute binding proteins (SBPs) facilitate the transport of the first reactant in a metabolic pathway, thereby constraining the regions of chemical space and the chemistries that must be considered for pathway reconstruction. We describe high-throughput protein production and differential scanning fluorimetry platforms, which enabled the screening of 158 SBPs against a 189 component library specifically tailored for this class of proteins. Like all screening efforts, this approach is limited by the practical constraints imposed by construction of the library, i.e., we can study only those metabolites that are known to exist and which can be made in sufficient quantities for experimentation. To move beyond these inherent limitations, we illustrate the promise of crystallographic- and mass spectrometric-based approaches for the unbiased use of entire metabolomes as screening libraries. Together, our approaches identified 40 new SBP ligands, generated experiment-based annotations for 2084 SBPs in 71 isofunctional clusters, and defined numerous metabolic pathways, including novel catabolic pathways for the utilization of ethanolamine as sole nitrogen source and the use of d-Ala-d-Ala as sole carbon source. These efforts begin to define an integrated strategy for realizing the full value of amassing genome sequence data.

Identification of the in vivo function of the high-efficiency D-mannonate dehydratase in Caulobacter crescentus NA1000 from the enolase superfamily.

The d-mannonate dehydratase (ManD) subgroup of the enolase superfamily contains members with varying catalytic activities (high-efficiency, low-efficiency, or no activity) that dehydrate d-mannonate and/or d-gluconate to 2-keto-3-deoxy-d-gluconate [Wichelecki, D. J., et al. (2014) Biochemistry 53, 2722-2731]. Despite extensive in vitro characterization, the in vivo physiological role of a ManD has yet to be established. In this study, we report the in vivo functional characterization of a high-efficiency ManD from Caulobacter crescentus NA1000 (UniProt entry B8GZZ7) by in vivo discovery of its essential role in d-glucuronate metabolism. This in vivo functional annotation may be extended to ~50 additional proteins.

Enzymatic and structural characterization of rTSγ provides insights into the function of rTSß.

In humans, the gene encoding a reverse thymidylate synthase (rTS) is transcribed in the reverse direction of the gene encoding thymidylate synthase (TS) that is involved in DNA biosynthesis. Three isoforms are found: α, ß, and γ, with the transcript of the α-isoform overlapping with that of TS. rTSß has been of interest since the discovery of its overexpression in methotrexate and 5-fluorouracil resistant cell lines. Despite more than 20 years of study, none of the rTS isoforms have been biochemically or structurally characterized. In this study, we identified rTSγ as an l-fuconate dehydratase and determined its high-resolution crystal structure. Our data provide an explanation for the observed difference in enzymatic activities between rTSß and rTSγ, enabling more informed proposals for the possible function of rTSß in chemotherapeutic resistance.

Investigating the physiological roles of low-efficiency D-mannonate and D-gluconate dehydratases in the enolase superfamily: pathways for the catabolism of L-gulonate and L-idonate.

The sequence/function space in the D-mannonate dehydratase subgroup (ManD) of the enolase superfamily was investigated to determine how enzymatic function diverges as sequence identity decreases [Wichelecki, D. J., et al. (2014) Biochemistry 53, 2722-2731]. That study revealed that members of the ManD subgroup vary in substrate specificity and catalytic efficiency: high-efficiency (kcat/KM = 10(3)-10(4) M(-1) s(-1)) for dehydration of D-mannonate, low-efficiency (kcat/KM = 10-10(2) M(-1) s(-1)) for dehydration of D-mannonate and/or D-gluconate, and no activity. Characterization of high-efficiency members revealed that these are ManDs in the D-glucuronate catabolic pathway {analogues of UxuA [Wichelecki, D. J., et al. (2014) Biochemistry 53, 4087-4089]}. However, the genomes of organisms that encode low-efficiency members of the ManDs subgroup encode UxuAs; therefore, these must have divergent physiological functions. In this study, we investigated the physiological functions of three low-efficiency members of the ManD subgroup and identified a novel physiologically relevant pathway for L-gulonate catabolism in Chromohalobacter salexigens DSM3043 as well as cryptic pathways for L-gulonate catabolism in Escherichia coli CFT073 and L-idonate catabolism in Salmonella enterica subsp. enterica serovar Enteritidis str. P125109. However, we could not identify physiological roles for the low-efficiency members of the ManD subgroup, allowing the suggestion that these pathways may be either evolutionary relics or the starting points for new metabolic potential.

Discovery of function in the enolase superfamily: D-mannonate and d-gluconate dehydratases in the D-mannonate dehydratase subgroup.

The continued increase in the size of the protein sequence databases as a result of advances in genome sequencing technology is overwhelming the ability to perform experimental characterization of function. Consequently, functions are assigned to the vast majority of proteins via automated, homology-based methods, with the result that as many as 50% are incorrectly annotated or unannotated ( Schnoes et al. PLoS Comput. Biol. 2009 , 5 ( 12 ), e1000605 ). This manuscript describes a study of the D-mannonate dehydratase (ManD) subgroup of the enolase superfamily (ENS) to investigate how function diverges as sequence diverges. Previously, one member of the subgroup had been experimentally characterized as ManD [dehydration of D-mannonate to 2-keto-3-deoxy-D-mannonate (equivalently, 2-keto-3-deoxy-D-gluconate)]. In this study, 42 additional members were characterized to sample sequence-function space in the ManD subgroup. These were found to differ in both catalytic efficiency and substrate specificity: (1) high efficiency (kcat/KM = 10(3) to 10(4) M(-1) s(-1)) for dehydration of D-mannonate, (2) low efficiency (kcat/KM = 10(1) to 10(2) M(-1) s(-1)) for dehydration of d-mannonate and/or D-gluconate, and 3) no-activity with either D-mannonate or D-gluconate (or any other acid sugar tested). Thus, the ManD subgroup is not isofunctional and includes D-gluconate dehydratases (GlcDs) that are divergent from the GlcDs that have been characterized in the mandelate racemase subgroup of the ENS (Lamble et al. FEBS Lett. 2004 , 576 , 133 - 136 ) (Ahmed et al. Biochem. J. 2005 , 390 , 529 - 540 ). These observations signal caution for functional assignment based on sequence homology and lay the foundation for the studies of the physiological functions of the GlcDs and the promiscuous ManDs/GlcDs.

Prediction of enzymatic pathways by integrative pathway mapping.

The functions of most proteins are yet to be determined. The function of an enzyme is often defined by its interacting partners, including its substrate and product, and its role in larger metabolic networks. Here, we describe a computational method that predicts the functions of orphan enzymes by organizing them into a linear metabolic pathway. Given candidate enzyme and metabolite pathway members, this aim is achieved by finding those pathways that satisfy structural and network restraints implied by varied input information, including that from virtual screening, chemoinformatics, genomic context analysis, and ligand -binding experiments. We demonstrate this integrative pathway mapping method by predicting the L-gulonate catabolic pathway in Haemophilus influenzae Rd KW20. The prediction was subsequently validated experimentally by enzymology, crystallography, and metabolomics. Integrative pathway mapping by satisfaction of structural and network restraints is extensible to molecular networks in general and thus formally bridges the gap between structural biology and systems biology.

Detection of liposome lysis utilizing an enzyme-substrate system.

A novel optical reporter system was developed to verify encapsulation and subsequent release of a foreign molecule in liposomes. The protocol utilizes a single enzyme and substrate. We encapsulate o-nitrophenyl-ß,D: -galactopyranoside (ONPG) and measure its release by detecting the levels of o-nitrophenol created when the encapsulated ONPG is released and hydrolyzed by ß-galactosidase. Using this method, liposome formation and subsequent lysis with Triton X-100 were verified. This new protocol eliminates the complications of multiple reaction enzyme detection methods, along with the chance for false negatives and unreliable data seen when using fluorescent particles as reporters.