An Atlas of the Thioredoxin Fold Class Reveals the Complexity of Function-Enabling Adaptations

The group of proteins that contain a thioredoxin (Trx) fold is huge and diverse. Assessment of the variation in catalytic machinery of Trx fold proteins is essential in providing a foundation for understanding their functional diversity and predicting the function of the many uncharacterized members of the class. The proteins of the Trx fold class retain common features—including variations on a dithiol CxxC active site motif—that lead to delivery of function. We use protein similarity networks to guide an analysis of how structural and sequence motifs track with catalytic function and taxonomic categories for 4,082 representative sequences spanning the known superfamilies of the Trx fold. Domain structure in the fold class is varied and modular, with 2.8% of sequences containing more than one Trx fold domain. Most member proteins are bacterial. The fold class exhibits many modifications to the CxxC active site motif—only 56.8% of proteins have both cysteines, and no functional groupings have absolute conservation of the expected catalytic motif. Only a small fraction of Trx fold sequences have been functionally characterized. This work provides a global view of the complex distribution of domains and catalytic machinery throughout the fold class, showing that each superfamily contains remnants of the CxxC active site. The unifying context provided by this work can guide the comparison of members of different Trx fold superfamilies to gain insight about their structure-function relationships, illustrated here with the thioredoxins and peroxiredoxins

Dual gradient-echo MRI of post-contraction changes in skeletal muscle blood volume and oxygenation

Analysis of post-contraction MRI signal intensity transients may allow non-invasive studies of microvascular reactivity and blood oxygenation recovery. The purpose of this study was to determine the physiological basis for post-contraction changes in short- (6 ms) and long- (46 ms) echo gradient-echo MRI signals (S(6) and S(46), respectively). Six healthy subjects were studied using dual gradient-echo MRI and near-infrared spectroscopy. S(6), S(46), total hemoglobin concentration ([THb]), and oxyhemoglobin saturation (%HbO(2)) were measured before, during, and after 1) 2 and 8 s dorsiflexion maximal voluntary contractions and 2) 5 min. of proximal arterial occlusion. Changes in S(6) and [THb] after 2 s contractions were similar to those following 8 s contractions, but changes in %HbO(2) and S(46) were greater following 8 s contractions than following 2 s contractions. [THb] and S(6) did not change during and following 5 minutes of arterial occlusion, but %HbO(2) and S(46) were both significantly depressed at similar occlusion durations. Also, distance measures indicated similarity between S(6) and [THb] and between S(46) and %HbO(2). We conclude that following brief human skeletal muscle contractions, changes in S(6) primarily reflect changes in blood volume and changes in S(46) primarily reflect changes in blood oxygenation

A glutathione transferase from Agrobacterium tumefaciens reveals a novel class of bacterial GST superfamily.

In the present work, we report a novel class of glutathione transferases (GSTs) originated from the pathogenic soil bacterium Agrobacterium tumefaciens C58, with structural and catalytic properties not observed previously in prokaryotic and eukaryotic GST isoenzymes. A GST-like sequence from A. tumefaciens C58 (Atu3701) with low similarity to other characterized GST family of enzymes was identified. Phylogenetic analysis showed that it belongs to a distinct GST class not previously described and restricted only in soil bacteria, called the Eta class (H). This enzyme (designated as AtuGSTH1-1) was cloned and expressed in E. coli and its structural and catalytic properties were investigated. Functional analysis showed that AtuGSTH1-1 exhibits significant transferase activity against the common substrates aryl halides, as well as very high peroxidase activity towards organic hydroperoxides. The crystal structure of AtuGSTH1-1 was determined at 1.4 Å resolution in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH). Although AtuGSTH1-1 adopts the canonical GST fold, sequence and structural characteristics distinct from previously characterized GSTs were identified. The absence of the classic catalytic essential residues (Tyr, Ser, Cys) distinguishes AtuGSTH1-1 from all other cytosolic GSTs of known structure and function. Site-directed mutagenesis showed that instead of the classic catalytic residues, an Arg residue (Arg34), an electron-sharing network, and a bridge of a network of water molecules may form the basis of the catalytic mechanism. Comparative sequence analysis, structural information, and site-directed mutagenesis in combination with kinetic analysis showed that Phe22, Ser25, and Arg187 are additional important residues for the enzyme's catalytic efficiency and specificity