Macro-to-Micro Structural Proteomics: Native Source Proteins for High-Throughput Crystallization

Totir, Monica A.; Nanao, Max H.; Gee, Christine L.; Moskaleva, Alisa; Gradia, Scott; Iavarone, Anthony T.; May, Andrew P.; Zubieta, Chloé; Alber, Tom

doi:10.1371/journal.pone.0032498

Cited by 38 publications

(45 citation statements)

References 49 publications

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“…Hydrolysis of the ␤-glycosidic bond is carried out by a catalytic mechanism that retains the conformation of the anomeric carbon; two conserved glutamate residues located in ␤ strands 4 and 7 acting as nucleophile and proton donor (10). In addition, several other aromatic residues in these enzymes are conserved and proposed to participate in substrate transport and/or binding (6,7). However, even minor changes in the substrate structure can bring about large alterations in enzymatic behavior.…”

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confidence: 99%

Characterization of a Novel Metagenome-Derived 6-Phospho-β-Glucosidase from Black Liquor Sediment

Yang

Niu

et al. 2013

Appl Environ Microbiol

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The enzyme 6-phospho-␤-glucosidase is an important member of the glycoside hydrolase family 1 (GH1). However, its catalytic mechanisms, especially the key residues determining substrate specificity and affinity, are poorly understood. A metagenomederived gene sequence, encoding a novel 6-phospho-␤-glucosidase designated Pbgl25-217, was isolated and characterized. The optimal conditions for enzymatic activity were 37°C and pH 7; Ca 2؉ , Mg 2؉ , and Mn 2؉ stabilized the activity of Pbgl25-217, whereas Ni 2؉ , Fe 2؉ , Zn 2؉ , Cu 2؉ , and Fe 3؉ inhibited its activity. The K m and V max of Pbgl25-217 were 4.8 mM and 1,987.0 U mg ؊1 , respectively. Seven conserved residues were recognized by multiple alignments and were tested by site-directed mutagenesis for their functions in substrate recognition and catalytic reaction. The results suggest that residues S427, Lys435, and Tyr437 act as "gatekeepers" in a phosphate-binding loop and play important roles in phosphate recognition. This functional identification may provide insights into the specificity of 6-phospho-␤-glycosidases in GH1 and be useful for designing further directed evolution.

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confidence: 99%

Characterization of a Novel Metagenome-Derived 6-Phospho-β-Glucosidase from Black Liquor Sediment

Yang

Niu

et al. 2013

Appl Environ Microbiol

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“…The crystal structure of wild-type BglA was obtained from the Protein Data Bank (PDB) (identification [ID] code 2XHY) (8), and mutant BglA(C195G) was modeled based on the crystal structure using the Rosetta software program (12). For both proteins, the centroid of the binding site of the sugar molecules was approximated using the average of the coordinates of the two catalytic glutamate residues (180 and 377).…”

Section: Methodsmentioning

confidence: 99%

“…Transformation of the parent strain with a plasmid carrying the bglA gene from the Sal ϩ mutant resulted in an Sal ϩ phenotype ( Table 2), indicating that the mutant carries an alteration within Arrows show the two active-site glutamate residues at position 180, which is a potential proton donor, and at 377, which is a hypothetical nucleophile site, based on sequence similarity (8). bglA.…”

Section: Isolation Of Salmentioning

confidence: 99%

“…The structure of the wild-type protein is based on PDB ID 2XHY (8), and the structure of mutant BglA was modeled using the Rosetta software with 2XHY as the template. The letter A indicates the approximate substrate binding pocket.…”

Section: Figmentioning

confidence: 99%

“…coli carries a constitutively expressed bglA gene encoding phospho-␤-glucosidase A, which is a part of the glycosyl hydrolase family 1 (8) and can hydrolyze phosphorylated arbutin, para-nitrophenyl-␤-glucoside, and phenyl ␤-glucoside but not phosphosalicin (9). However, wild-type strains are unable to utilize arbutin because of the lack of a permease (9).…”

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confidence: 99%

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Evolution of Aromatic β-Glucoside Utilization by Successive Mutational Steps in Escherichia coli

2015

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The bglA gene of Escherichia coli encodes phospho-␤-glucosidase A capable of hydrolyzing the plant-derived aromatic ␤-glucoside arbutin. We report that the sequential accumulation of mutations in bglA can confer the ability to hydrolyze the related aromatic ␤-glucosides esculin and salicin in two steps. In the first step, esculin hydrolysis is achieved through the acquisition of a four-nucleotide insertion within the promoter of the bglA gene, resulting in enhanced steady-state levels of the bglA transcript. In the second step, hydrolysis of salicin is achieved through the acquisition of a point mutation within the bglA structural gene close to the active site without the loss of the original catabolic activity against arbutin. These studies underscore the ability of microorganisms to evolve additional metabolic capabilities by mutational modification of preexisting genetic systems under selection pressure, thereby expanding their repertoire of utilizable substrates. The extraordinary versatility and adaptability exhibited by microorganisms are primarily responsible for their success in occupying a broad spectrum of niches. Bacteria have evolved different strategies to cope with a variety of stresses encountered in their natural habitats. When forced to grow in the presence of a novel substrate as the sole carbon source, bacteria are able to evolve the capability to utilize the novel substrate by modifying preexisting genetic systems having related functions (1, 2).Escherichia coli has several catabolic systems for the utilization of plant-derived aromatic ␤-glucosides such as arbutin (Arb), salicin (Sal), and esculin (Esc) (Fig. 1) that encode a sugar-specific permease that is a part of the phosphotransferase system (PTS) and a phospho-␤-glucosidase necessary for the hydrolysis of the sugar. Interestingly, many of these genetic systems are silent and need a step of mutational activation to be functional. The silent bgl operon of E. coli enables the utilization of arbutin and salicin upon mutational activation (3). A homologue of the bgl operon in Erwinia has been shown to confer the ability to utilize esculin in addition to arbutin and salicin (4). Prompted by this observation, el Hassouni et al. have examined the role of the bgl operon of E. coli in esculin utilization. They have reported that Bgl ϩ strains of E. coli can hydrolyze esculin and that this ability is lost in mutants in which the transport and hydrolysis functions have been abrogated (4).The bgl operon consists primarily of three genes, bglG, bglF, and bglB, encoding a positive regulator that acts as an antiterminator of transcription, the permease that transports and concomitantly phosphorylates arbutin and salicin, and the phospho-␤-glucosidase B that hydrolyzes phospho-arbutin and phospho-salicin, respectively. The bgl operon is silent in most wild-type (WT) strains due to the presence of negative elements upstream of the promoter; mutations such as the transposition of insertion elements that abrogate the negative elements result in the activa...

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Prediction of chaperonin GroE substrates using small structural patterns of proteins

et al. 2023

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Molecular chaperones are indispensable proteins that assist the folding of aggregation‐prone proteins into their functional native states, thereby maintaining organized cellular systems. Two of the best‐characterized chaperones are the Escherichia coli chaperonins GroEL and GroES (GroE), for which in vivo obligate substrates have been identified by proteome‐wide experiments. These substrates comprise various proteins but exhibit remarkable structural features. They include a number of α/β proteins, particularly those adopting the TIM β/α barrel fold. This observation led us to speculate that GroE obligate substrates share a structural motif. Based on this hypothesis, we exhaustively compared substrate structures with the MICAN alignment tool, which detects common structural patterns while ignoring the connectivity or orientation of secondary structural elements. We selected four (or five) substructures with hydrophobic indices that were mostly included in substrates and excluded in others, and developed a GroE obligate substrate discriminator. The substructures are structurally similar and superimposable on the 2‐layer 2α4β sandwich, the most popular protein substructure, implying that targeting this structural pattern is a useful strategy for GroE to assist numerous proteins. Seventeen false positives predicted by our methods were experimentally examined using GroE‐depleted cells, and 9 proteins were confirmed to be novel GroE obligate substrates. Together, these results demonstrate the utility of our common substructure hypothesis and prediction method.

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Macro-to-Micro Structural Proteomics: Native Source Proteins for High-Throughput Crystallization

Cited by 38 publications

References 49 publications

Characterization of a Novel Metagenome-Derived 6-Phospho-β-Glucosidase from Black Liquor Sediment

Characterization of a Novel Metagenome-Derived 6-Phospho-β-Glucosidase from Black Liquor Sediment

Evolution of Aromatic β-Glucoside Utilization by Successive Mutational Steps in Escherichia coli

Prediction of chaperonin GroE substrates using small structural patterns of proteins

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