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The soil bacterium Rhodococcus jostii RHA1 contains two dye-decolorizing peroxidases (DyPs) named according to the subfamily they represent: DypA, predicted to be periplasmic, and DypB, implicated in lignin degradation. Steady-state kinetic studies of these enzymes revealed that they have much lower peroxidase activities than C- and D-type DyPs. Nevertheless, DypA showed 6-fold greater apparent specificity for the anthraquinone dye Reactive Blue 4 (k(cat)/K(m) = 12800 ± 600 M(-1) s(-1)) than either ABTS or pyrogallol, consistent with previously characterized DyPs. By contrast, DypB showed the greatest apparent specificity for ABTS (k(cat)/K(m) = 2000 ± 100 M(-1) s(-1)) and also oxidized Mn(II) (k(cat)/K(m) = 25.1 ± 0.1 M(-1) s(-1)). Further differences were detected using electron paramagnetic resonance (EPR) spectroscopy: while both DyPs contained high-spin (S = (5)/(2)) Fe(III) in the resting state, DypA had a rhombic high-spin signal (g(y) = 6.32, g(x) = 5.45, and g(z) = 1.97) while DypB had a predominantly axial signal (g(y) = 6.09, g(x) = 5.45, and g(z) = 1.99). Moreover, DypA reacted with H(2)O(2) to generate an intermediate with features of compound II (Fe(IV)═O). By contrast, DypB reacted with H(2)O(2) with a second-order rate constant of (1.79 ± 0.06) × 10(5) M(-1) s(-1) to generate a relatively stable green-colored intermediate (t(1/2) ∼ 9 min). While the electron absorption spectrum of this intermediate was similar to that of compound I of plant-type peroxidases, its EPR spectrum was more consistent with a poorly coupled protein-based radical than with an [Fe(IV)═O Por(•)](+) species. The X-ray crystal structure of DypB, determined to 1.4 Å resolution, revealed a hexacoordinated heme iron with histidine and a solvent species occupying axial positions. A solvent channel potentially provides access to the distal face of the heme for H(2)O(2). A shallow pocket exposes heme propionates to the solvent and contains a cluster of acidic residues that potentially bind Mn(II). Insight into the structure and function of DypB facilitates its engineering for the improved degradation of lignocellulose.

SummaryIron is critical for virtually all forms of life. The production of high-affinity iron chelators, siderophores, and the subsequent uptake of iron-siderophore complexes are a common strategy employed by microorganisms to acquire iron. Staphylococcus aureus produces siderophores but genetic information underlying their synthesis and transport is limited. Previous work implicated the sbn operon in siderophore synthesis and the sirABC operon in uptake. Here we characterize a second siderophore biosynthetic locus in S. aureus; the locus consists of four genes (in strain Newman these open reading frames are designated NWMN_2079-2082) which, together, are responsible for the synthesis and export of staphyloferrin A, a polycarboxylate siderophore. While deletion of the NWMN_2079-2082 locus did not affect iron-restricted growth of S. aureus, strains bearing combined sbn and NWMN_2079-2082 locus deletions produced no detectable siderophore and demonstrated severely attenuated iron-restricted growth. Adjacent to NWMN_2079-2082 resides the htsABC operon, encoding an ABC transporter previously implicated in haem acquisition. We provide evidence here that HtsABC, along with the FhuC ATPase, is required for the uptake of staphyloferrin A. The crystal structure of apo-HtsA was determined and identified a large positively charged region in the substrate-binding pocket, in agreement with a role in binding of anionic staphyloferrin A.

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The T box leader sequence is an RNA element that controls gene expression by binding directly to a specific tRNA and sensing its aminoacylation state. This interaction controls expression of amino acid-related genes in a negative feedback loop. The T box RNA structure is highly conserved, but its tRNA binding mechanism is only partially understood. Known sequence elements are the specifier sequence, which recognizes the tRNA anticodon, and the antiterminator bulge, which base pairs with the tRNA acceptor end. Here, we reveal the crucial function of the highly conserved stem I distal region in tRNA recognition and report its 2.65-Å crystal structure. The apex of this region contains an intricately woven looploop interaction between two conserved motifs, the Adenine-guanine (AG) bulge and the distal loop. This loop-loop structure presents a base triple on its surface that is optimally positioned for basestacking interactions. Mutagenesis, cross-linking, and small-angle X-ray scattering data demonstrate that the apical base triple serves as a binding platform to dock the tRNA D-and T-loops. Strikingly, the binding platform strongly resembles the D-and T-loop binding elements from RNase P and the ribosome exit site, suggesting that this loop-loop structure may represent a widespread tRNA recognition platform. We propose a two-checkpoint molecular ruler model for tRNA decoding in which the information content of tRNA is first examined through specifier sequence-anticodon interaction, and the length of the tRNA anticodon arm is then measured by the distal loop-loop platform. When both conditions are met, tRNA is secured, and its aminoacylation state is sensed.riboswitch | RNA-RNA complex C ells must maintain appropriate intracellular pools of aminoacylated transfer RNA (aa-tRNA) to survive. This is usually accomplished by tight regulation of many cellular factors, including amino acid biosynthesis and transporter genes, and tRNA synthetases (1). Most Gram-positive bacteria use the T box regulatory system to control their aa-tRNA levels (2). T box elements are a special family of regulatory RNAs located in the 5′-untranslated region of the mRNA for a regulated gene or operon. Unlike small molecule-sensing riboswitches, T box RNAs recognize tRNAs containing the cognate anticodon and discriminate between uncharged tRNA and aa-tRNA. They usually control gene expression via premature transcription termination, although there are rare examples that are predicted to regulate at the level of translation initiation (2-4). The T box motif was initially identified in the Bacillus subtilis (Bsub) tyrosyl-tRNA synthetase gene (tyrS) and was shown to respond to the aminoacylation state of tyrosine tRNA (5, 6). More than 1,000 T box elements have since been identified (3, 4).Selective tRNA recognition is achieved through a collective effort from several conserved domains in the T box RNA [Geobacillus kaustophilus (Gkau) glyQS T box sequence is shown in Fig. 1]. Stem I is the largest element. It is an ∼100-nucleotide (nt) stem-loop s...