Pyruvate methyl proton exchange rate of the wild type and variants of the HMG/CHA aldolase in the absence and presence of P_<i>i</i>.

<p>Pyruvate methyl proton exchange rate of the wild type and variants of the HMG/CHA aldolase in the absence and presence of P_<i>i</i>.</p

P_<i>i</i> activation with different substrates.

<p>P_<i>i</i> activation with different substrates.</p

Investigation into the Mode of Phosphate Activation in the 4-Hydroxy-4-Methyl-2-Oxoglutarate/4-Carboxy-4-Hydroxy-2-Oxoadipate Aldolase from <i>Pseudomonas putida</i> F1

<div><p>The 4-hydroxy-4-methyl-2-oxoglutarate (HMG)/4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolase is the last enzyme of both the gallate and protocatechuate 4,5-cleavage pathways which links aromatic catabolism to central cellular metabolism. The enzyme is a class II, divalent metal dependent, aldolase which is activated in the presence of inorganic phosphate (P_<i>i</i>), increasing its turnover rate >10-fold. This phosphate activation is unique for a class II aldolase. The aldolase pyruvate methyl proton exchange rate, a probe of the general acid half reaction, was increased 300-fold in the presence of 1 mM P_<i>i</i> and the rate enhancement followed saturation kinetics giving rise to a <i>K</i>_M of 397 ± 30 μM. Docking studies revealed a potential P_<i>i</i> binding site close to, or overlapping with, the proposed general acid water site. Putative P_<i>i</i> binding residues were substituted by site-directed mutagenesis which resulted in reductions of P_<i>i</i> activation. Significantly, the active site residue Arg-123, known to be critical for the catalytic mechanism of the enzyme, was also implicated in supporting P_<i>i</i> mediated activation.</p></div

Docking of P_<i>i</i> into the active site of the HMG/CHA aldolase.

<p>The HMG/CHA aldolase as represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164556#pone.0164556.g001" target="_blank">Fig 1</a>. (A) The center of mass of each P_<i>i</i> ion is shown as an orange sphere and the grouped putative binding sites are numbered. A representative binding mode for sites 1 (B), 2 (C), and 3 (D) are shown with the native structures residues as lines with significant positional changes indicated with arrows. Potential hydrogen bonds between P_<i>i</i> and the protein, defined by distances between 2.8 and 3.2 Å, are indicated by dashes.</p

Comparison of the HMG/CHA aldolase with the PT proteins.

<p>(A) Overall structural comparison with the proteins oriented via structural alignment of the conserved domain which is coloured white with arrows pointing out the aldolase Arg-123 and transferase PH residue. The HMG/CHA aldolases Mg²⁺ and pyruvate are shown as a green sphere and yellow sticks, respectively. EI:PTS from <i>E</i>. <i>coli</i> (PDB: 2HWG) is shown with its PEP binding domain coloured red its and helical bundle, which supports HPr binding, coloured green. The EI:PTS Mg²⁺ and oxalate are shown as a green sphere and yellow sticks, respectively. PK from <i>Geobacillus strearothermophilus</i> (PDB: 2E28) is shown with its kinase domain coloured magenta its and effector domain coloured dark green. The PPDK from Maize (PDB: 1VBH) is shown with its nucleotide binding domain, linker region, and PEP binding domain coloured in cyan, pink, and brown, respectively. The PPDK Mg²⁺ is shown as a green sphere and its bound PEP as yellow sticks. (B) Section of the primary sequences in the structural alignment indicating the HMG/CHA aldolases essential Arg-123 in a magenta box, the PH with an orange sphere, and the PPDK threonine observed as a site of phosphorylation in plants as a green sphere. (C) Overlay of the HMG/CHA aldolase and EI:PTS with comparison of key residues. The enzymes are coloured as in panel A except, for clarity, the conserved domain in EI:PTS is coloured blue.</p

The HMG/CHA aldolase reactions and active site.

<p>(A) Aldol cleavage catalyzed by the HMG/CHA aldolase where for HMG the R group is -CH₃ which yields 2 moles of pyruvate and for CHA the R group is -CH₂COO^- yielding 1 mole each of pyruvate and oxaloacetate. (B) Oxaloacetate decarboxylase reaction catalyzed by the aldolase producing either carbon dioxide or bicarbonate. (C) Active site of the aldolase showing the pyruvate carbon in yellow sticks, the bound magnesium ion as a green sphere, and key water molecules as red spheres. The structural representation was generated in Pymol (version 1.7) using atomic coordinates of the HMG/CHA aldolase (PDB ID: 3NOJ).</p

Biochemical and Structural Analysis of RraA Proteins To Decipher Their Relationships with 4‑Hydroxy-4-methyl-2-oxoglutarate/4-Carboxy-4-hydroxy-2-oxoadipate Aldolases

4-Hydroxy-4-methyl-2-oxoglutarate (HMG)/4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolases are class II (divalent metal ion dependent) pyruvate aldolases from the <i>meta</i> cleavage pathways of protocatechuate and gallate. The enzyme from <i>Pseudomonas putida</i> F1 is structurally similar to a group of proteins termed regulators of RNase E activity A (RraA) that bind to the regulatory domain of RNase E and inhibit the ribonuclease activity in certain bacteria. Analysis of homologous RraA-like proteins from varying species revealed that they share sequence conservation within the active site of HMG/CHA aldolase. In particular, the <i>P. putida</i> F1 HMG/CHA aldolase has a D-X₂₀-R-D motif, whereas a G-X₂₀-R-D-X₂-E/D motif is observed in the structures of the RraA-like proteins from <i>Thermus thermophilus</i> HB8 (<i>Tt</i>RraA) and <i>Saccharomyces cerevisiae</i> S288C (Yer010Cp) that may support metal binding. <i>Tt</i>RraA and Yer010Cp were found to contain HMG aldolase and oxaloacetate decarboxylase activities. Similar to the <i>P. putida</i> F1 HMG/CHA aldolase, both <i>Tt</i>RraA and Yer010Cp enzymes required divalent metal ions for activity and were competitively inhibited by oxalate, a pyruvate enolate analogue, suggesting a common mechanism among the enzymes. The RraA from <i>Escherichia coli</i> (<i>Ec</i>RraA) lacked detectable C–C lyase activity. Upon restoration of the G-X₂₀-R-D-X₂-E/D motif, by site-specific mutagenesis, the <i>Ec</i>RraA variant was able to catalyze oxaloacetate decarboxylation. Sequence analysis of RraA-like gene products found across all the domains of life revealed conservation of the metal binding motifs that can likely support a divalent metal ion-dependent enzyme reaction either in addition to or in place of the putative RraA function

MOESM1 of Biochemical and structural features of diverse bacterial glucuronoyl esterases facilitating recalcitrant biomass conversion

Additional file 1: Table S1. Percent sequence identity and percent query coverage (in brackets) between all CE15 enzymes used in this study. Sequence identity values for CE15 enzymes within one organism are marked green (O. terrae), magenta (S. linguale) and blue (S. usitatus). The query sequences are presented in the top row.Kinetic parameters of O. terrae, S. linguale, and S. usitatus CE15 enzymes on model. Table S2. Kinetic parameters of O. terrae, S. linguale, and S. usitatus CE15 enzymes on model substrates. Esterase activity with benzyl (Bnz), allyl (Allyl), methyl (Me) esters of glucuronoate (GlcA) and galacturonoate (GalA) are shown in addition to acetyl esterase activity with 4-nitrophenol acetate (pNP-Ac) and 1,2,3,4-tetra-O-acetyl-β-d-xylopyranose (TetAcXyl). Table S3. Primers used for cloning CE15 constructs and for qPCR of S. linguale CE15 members. Table S4. Table of crystallographic statistics. Figure S1. Unrooted phylogenetic tree of all members of CE15 (catalytic domains), with Genbank accession numbers as identifiers. Yellow branches represent fungal members, circles indicate biochemically characterized members, and stars represent members with solved structures. Targets of this study are shown using the same color code as in the main text: green for O. terrae, red for S. linguale, and blue for S. usitatus. Figure S2. Model substrates used in this study: (A) BnzGlcA, (B) AllylGlcA, (C) MeGlcA, (D) MeGalA, (E) pNP-Ac and (F) TetAcXyl. Figure S3. Effect of pH on BnzGlcA esterase activity for CE15 enzymes from O. terrae (OtCE15 A-D, panels A-D), S. linguale (SlCE15 A-C, panels E-G), and S. usitatus (SuCE15 A-C, panels H-J). Mean values of relative activity from duplicate measurements are plotted with standard error of the mean. Figure S4. Structure-based sequence alignment of all CE15 enzymes structurally characterized to date. Similar residues are written in red text while conserved residues are written in white text over a red background. The insertion regions found in the bacterial structures relative to the fungal counterparts are highlighted in yellow. The residues of the canonical catalytic triad are indicated by cyan arrows below the text. The aspartate in MZ0003 proposed to act as the acidic residue of the catalytic triad, in place of the missing canonical glutamate, is indicate by a black arrow below the text. Note that both OtCE15A and SuCE15C also have an aspartate at the same position while additionally having the glutamate of the canonical catalytic triad. Residues hydrogen bonding with 4-O-methyl-glucuronoate in the StGE2 co-crystal structure are indicated by blue arrows above the text. The isoleucine and leucine comprising a hydrophobic patch near the 4-O-methyl substituent in the StGE2 co-crystal structure are indicated by magenta arrows. The phenylalanine conserved in the bacterial structures possibly aiding in positioning in aromatic substituents of the sugar esters is indicated with a grey arrow. The disulfide bridges formed in the fungal structures are indicated above the alignment by numbering in green text. Figure S5. Multiple sequence alignment of characterized glucuronoyl esterases. Similar residues are written in red text while conserved residues are written in white text over a red background. The insertion regions found in the bacterial structures relative to the fungal counterparts are highlighted in yellow. The residues of the conserved catalytic triad are colored cyan. Note that glutamate of the catalytic triad is not conserved in all bacterial esterases and the position of the equivalent acidic residue in MZ0003 is also colored cyan. Arrows indicating significant residues are colored as in Additional file 5: Figure S4. Figure S6. Growth curves of S. linguale when grown with different additives or on different carbon sources. S. linguale did not grow on standard minimal media and an optimized media for bacterial growth was determined experimentally (see methods for formulation). (A) Growth curves of S. linguale in the optimized media without a carbon source (red), with 0.3% (w/v) glucose (blue) and in the media containing glucose but in the absence of either trace metals and vitamins (green), sodium phosphate pH 7.5 (magenta), or magnesium sulphate (cyan). (B) Growth of S. linguale in optimized media with 0.3% of either glucose (blue), xylose (purple), or xylan from corn cob (yellow)