Kinetic parameters of wild-type and mutant Alr_<i>Tt</i>.

<p>Kinetic parameters of wild-type and mutant Alr_<i>Tt</i>.</p

Additional file 1 of Gitelman syndrome with Graves’ disease leading to rhabdomyolysis: a case report and literature review

Supplementary Material

Crystal Structure of a Thermostable Alanine Racemase from <i>Thermoanaerobacter tengcongensis</i> MB4 Reveals the Role of Gln360 in Substrate Selection

<div><p>Pyridoxal 5’-phosphate (PLP) dependent alanine racemase catalyzes racemization of L-Ala to D-Ala, a key component of the peptidoglycan network in bacterial cell wall. It has been extensively studied as an important antimicrobial drug target due to its restriction in eukaryotes. However, many marketed alanine racemase inhibitors also act on eukaryotic PLP-dependent enzymes and cause side effects. A thermostable alanine racemase (Alr_Tt) from <i>Thermoanaerobacter tengcongensis</i> MB4 contains an evolutionarily non-conserved residue Gln360 in inner layer of the substrate entryway, which is supposed to be a key determinant in substrate specificity. Here we determined the crystal structure of Alr_Tt in complex with L-Ala at 2.7 Å resolution, and investigated the role of Gln360 by saturation mutagenesis and kinetic analysis. Compared to typical bacterial alanine racemase, presence of Gln360 and conformational changes of active site residues disrupted the hydrogen bonding interactions necessary for proper PLP immobilization, and decreased both the substrate affinity and turnover number of Alr_Tt. However, it could be complemented by introduction of hydrophobic amino acids at Gln360, through steric blocking and interactions with a hydrophobic patch near active site pocket. These observations explained the low racemase activity of Alr_Tt, revealed the essential role of Gln360 in substrate selection, and its preference for hydrophobic amino acids especially Tyr in bacterial alanine racemization. Our work will contribute new insights into the alanine racemization mechanism for antimicrobial drug development.</p></div

Overall crystal structure of Alr_<i>Tt</i>.

<p><b>(A)</b> Overall structure of Alr_<i>Tt</i> monomer. N-terminal α/β barrel domain, and the C terminal β-strand domain are shown in orange, phosphate group (red) and L-Ala (green) in the active site are shown in spheres. <b>(B)</b> Dimer of Alr_<i>Tt</i>, it is formed by two head-to-tail associated monomers (colored in orange and blue) in one asymmetric unit. The dimer interface is indicated by dashed box. The phosphate group (red) and L-Ala (green) are shown in sticks. <b>(C)</b> Comparison of the overall architectures of alanine racemase from a gram positive bacteria <i>Bacillus stearothermophilus</i> (Alr_<i>Bst</i>, PDB 1SFT, magenta), a gram negative bacteria <i>Pseudomonas aeruginosa</i> (DadX_<i>pao</i>, PDB 1RCQ, orange), and <i>Clostridium difficile</i> strain 630 (Alr_<i>Cd</i>, PDB 4LUS, green) with Alr_<i>Tt</i> (PDB 4Y2W, blue). The structures are superimposed at the N-terminal α/β barrel domain, the shift of the β-strand domain are represented by distance from Arg276 in Alr_<i>Tt</i> to corresponding residues like Arg261 in DadX_<i>pao</i>, Thr273 in Alr_<i>Bst</i> and Gly276 in Alr_<i>Cd</i>. <b>(D)</b> Hydrogen bonding interactions mediating the dimer formation, residues in N-terminal α/β barrel domain of one monomer (orange) and the C terminal β-strand domain of the another monomer (blue) are shown in sticks, the hydrogen bonds are indicated as dashed lines.</p

Relative activity of the saturation mutants of Gln360 in L-Ala racemization (A), and the relative activity of wild-type (B) and Q360Y mutant (C) in racemization of ten L-amino acids.

<p><b>(A)</b> The racemase activities of the saturation mutants of Gln360 relative to wild-type Alr_<i>Tt</i> are represented by columns with means ± SD of quadruplicate experiments. <b>(B)</b> The racemase activities of ten L-amino acids relative to L-Ala catalyzed by wild-type Alr_<i>Tt</i> are represented by columns with means ± SD of quadruplicate experiments. <b>(C)</b> The relative amino acids specificity of Q360Y towards ten L-amino acids is shown in same profile as <b>(B)</b>.</p

Structural based sequence alignment of Alr_<i>Tt</i>, DadX_<i>Tt</i> and other three representative bacterial alanine racemases.

<p>Amino acid sequences of alanine racemase from a gram positive bacteria <i>Bacillus stearothermophilus</i> (Alr_<i>Bst</i>), a gram negative bacteria <i>Pseudomonas aeruginosa</i> (DadX_<i>pao</i>), and <i>Clostridium difficile</i> strain 630 (Alr_<i>Cd</i>) are aligned with Alr_<i>Tt</i> and DadX_<i>Tt</i> from <i>T</i>. <i>tengcongensis</i> MB4. Amino acids are numbered and secondary structures are labeled, strictly conserved amino acids are highlighted in yellow box. Amino acids form the substrate entryway are colored in blue (middle layer) and magenta (inner layer), key catalytic residues mediating the phosphate group and L-Ala binding are colored in red, residues necessary for hydrogen bonding interactions for PLP-binding are colored in green. Two key catalytic residues Lys40 and Tyr268’ are marked with a star. The hydrophobic patch (Pro225-Arg337) in Alr_<i>Tt</i> is indicated by a red box.</p

Comparison of the active site pocket of Alr_<i>Tt</i> with other three bacterial alanine racemases.

<p><b>(A)</b> The active site pocket of Alr_<i>Tt</i> (PDB 4Y2W), phosphate group, L-Ala and key amino acids that involved in substrate binding are shown in sticks, the hydrogen bonding interactions are indicated. Same view of the active site pocket of other three bacteria alanine racemase in complex with substrates are shown in same profile: <b>(B)</b> the PLP-D-Ala complex of Alr_<i>Bst</i> (PDB 1L6G), <b>(C)</b> PLP complex of Alr_<i>Cd</i> (PDB 4LUS), <b>(D)</b> PLP and DLY complex of DadX_<i>pao</i> (PDB 1RCQ).</p

The hydrophobic patch and its interaction with active site pocket.

<p><b>(A)</b> The hydrophobic patch (Pro225-Arg237) in Alr_<i>Tt</i> (PDB 4Y2W) is colored in gray, the active site pocket is shown in orange, the substrate and key amino acids mediating the interactions are shown in sticks, the hydrogen bonding interactions are indicated. The hydrophobic patch and active site pocket of the other three bacterial alanine racemases are shown in same view: <b>(B)</b> the PLP-D-Ala complex of Alr_<i>Bst</i> (PDB 1L6G). <b>(C)</b> PLP complex of Alr_<i>Cd</i> (PDB 4LUS), <b>(D)</b> PLP and DLY complex of DadX_<i>pao</i> (PDB 1RCQ).</p

Additional file 2 of A case report and literature review of Carney complex with atrial adenomyxoma

Additional file 2: Figure S2. Postoperative thyroid ultrasound and breast ultrasound at follow-up. (A) Postoperative thyroid ultrasound showed a postoperative image of the right thyroid carcinoma. (B) Postoperative thyroid ultrasound showed the left thyroid lobe was negative. (C) Breast ultrasound at follow-up showed multiple hypoechoic nodules in the right breast, cord-like slightly hyperechoic band, maximum size of 19×6 mm, regular in shape, clear in boundary, no blood flow signal, with a BI-RADS classification of 3

Additional file 1 of A case report and literature review of Carney complex with atrial adenomyxoma

Additional file 1: Figure S1. Thyroid fine needle aspiration (FNA) showed a cell group with nuclear crowding, irregularities in the nuclear membrane, intranuclear inclusions and nuclear grooves, which indicated suspicious papillary thyroid carcinoma, Bethesda V