TCA Cycle Involved Enzymes SucA and Kgd, as well as MenD: Efficient Biocatalysts for Asymmetric C–C Bond Formation

Asymmetric mixed carboligation reactions of α-ketoglutarate with different aldehydes were explored with the thiamine diphosphate dependent enzymes SucA from <i>E. coli</i>, Kgd from <i>Mycobacterium tuberculosis</i>, and MenD from <i>E. coli</i>. All three enzymes proved to be efficient biocatalysts to selectively deliver chiral δ-hydroxy-γ-keto acids with moderate to excellent stereoselectivity. The high regioselectivity is due to the preserved role of α-ketoglutarate as acyl donor for these enzyme-catalyzed reactions

SoxR as a Single-Cell Biosensor for NADPH-Consuming Enzymes in <i>Escherichia coli</i>

An ultra-high-throughput screening system for NADPH-dependent enzymes, such as stereospecific alcohol dehydrogenases, was established. It is based on the [2Fe–2S] cluster-containing transcriptional regulator SoxR of Escherichia coli<i> </i>that activates expression of <i>soxS</i> in the oxidized but not in the reduced state of the cluster. As SoxR is kept in its reduced state by NADPH-dependent reductases, an increased NADPH demand of the cell counteracts SoxR reduction and increases <i>soxS</i> expression. We have taken advantage of these properties by placing the <i>eyfp</i> gene under the control of the <i>soxS</i> promoter and analyzed the response of E. coli cells expressing an NADPH-dependent alcohol dehydrogenase from Lactobacillus brevis (<i>Lb</i>Adh), which reduces methyl acetoacetate to (<i>R</i>)-methyl 3-hydroxybutyrate. Under suitable conditions, the specific fluorescence of the cells correlated with the substrate concentration added and with <i>Lb</i>Adh enzyme activity, supporting the NADPH responsiveness of the sensor. These properties enabled sorting of single cells harboring wild-type <i>Lb</i>Adh from those with lowered or without <i>Lb</i>Adh activity by fluorescence-activated cell sorting (FACS). In a proof-of-principle application, the system was used successfully to screen a mutant <i>Lb</i>Adh library for variants showing improved activity with the substrate 4-methyl-2-pentanone

Lipoprotein AmyE is triacylated.

<p><b>A.</b> AmyE localization in <i>C. glutamicum</i> wild-type cells expressing AmyE (pAmyE) or a variant of AmyE with a point mutation substituting the cysteine +1 by a leucine (pAmyE^C1L). An empty vector (pCGL482) was used as a control. Membrane and secreted proteins were analyzed by SDS-PAGE followed by immunoblotting using monoclonal anti-his antibodies. The band labeled with an asterisk corresponds to a shorter form of AmyE. <b>B.</b> AmyE (left panel) and AmyE^C1L (right panel) were purified and analyzed by SDS PAGE before (lane 1) or after Triton X114 extraction. Proteins from both aqueous (lane 2) and detergent (lane 3) phases were precipitated and loaded on the gel. <b>C.</b> MALDI mass measurements of intact purified AmyE and AmyE^C1L proteins. Estimated mass accuracy is 150 Da. <b>D.</b> MALDI PMFs of AmyE and AmyE^C1L proteins purified from <i>C. glutamicum</i> wild-type strain. The <i>m/z</i> 3200–4800 region of the mass spectra of AmyE and AmyE^C1L tryptic peptides after DDM/CHCl₃-CH₃OH treatment is shown and significant monoisotopic [M+H]⁺¹ peaks are indicated. Upper panel: <i>m/z</i> 4097.10 (bold) corresponds to the triacylated AmyE_1–29 peptide while <i>m/z</i> 3451.65, 3821.53 and 4045.88 match to internal tryptic peptides, AmyE_334–365, AmyE_366–397 and AmyE_55–91 respectively. <i>m/z</i> 4061.74 and 4077.54 peaks could correspond to mono- and di- oxidized AmyE_55–91 peptides. Bottom panel: <i>m/z</i> 3292.60 (bold) corresponds to the C1L-mutated AmyE_1–29 peptide while <i>m/z</i> 3451.65 and 3821.72 match to AmyE_334–365 and AmyE_366–397 peptides. The <i>m/z</i> 4061.79, 4077.80, 4093.86 and 4109.83 peaks match to mono-, di-, tri- and tetra-oxidized AmyE_55–91 peptides. The <i>m/z</i> 4342.89 peak corresponds to the di-oxidized AmyE_277–315 peptide. It’s worth noting that AmyE^C1L peptides were more often detected in the oxidized state than AmyE peptides. Insets aim at emphasizing the specificity of the <i>m/z</i> 4097.10 signal detected only in the AmyE spectrum. Asterisks indicate that <i>m/z</i> assignments are not accurate because of low mass resolution and weak signal/noise ratio. <b>E.</b> Sequence of the recombinant purified wild-type AmyE protein. Identified unmodified peptides after trypsin digestion and DDM/CHCl₃-CH₃OH treatment are shown in boldface type on the amino acid sequence of recombinant AmyE.</p

<i>M. tuberculosis</i> Ppm1 is active in <i>C. glutamicum.</i>

<p>Comparison of MALDI PMF profiles of LppX protein purified from Δ<i>ppm2</i> (pMt-<i>ppm1</i>) and Δ<i>ppm1</i> (pMt-<i>ppm1</i>). The <i>m/z</i> 3500–4550 region of the mass spectra of LppX tryptic peptides after DDM/CHCl₃-CH₃OH treatment is shown and significant monoisotopic [M+H]⁺¹ peaks are indicated. Upper spectrum: in the Δ<i>ppm2</i> (pMt-<i>ppm1</i>) <i>m/z</i> peaks corresponding to different glycosylated forms of the triacylated LppX_1–29 peptide were observed (<i>m/z</i> 3941.22, 4041.18, 4103.26, 4203.25, 4427.28 and 4527.23) as well as the peak of the non-glycosylated triacylated LppX_1–29 peptide (<i>m/z</i> 3779.20) Bottom spectrum: in the Δ<i>ppm1</i> (pMt-<i>ppm1</i>) strain, peaks corresponding to different glycosylated forms of the triacylated LppX_1–29 peptide were observed (<i>m/z</i> 4103.22, 4203.26, 4427.18 and 4527.12, in bold). Three peaks (<i>m/z</i> 3547.83, 3662.78 and 3943.95) were detected but not identified. • = 1 hexose (Δm = 162 Da). • = unknown modification (Δm = 262 Da). “ni” means not identified and asterisks indicate that <i>m/z</i> assignments are not very accurate.</p

Cg-Ppm1 is required for LppX glycosylation.

<p>Comparison of MALDI PMF profiles of LppX protein purified from <i>C. glutamicum</i> wild-type and Δ<i>ppm1</i> strains. The <i>m/z</i> 3500–4550 region of the mass spectra of LppX tryptic peptides after DDM/CHCl₃-CH₃OH treatment is shown and significant monoisotopic [M+H]⁺¹ peaks are annotated. Upper panel: <i>m/z</i> peaks correspond to glycosylated and triacylated LppX_1–29 peptides are indicated in bold (<i>m/z</i> 4103.09, 4203.03, 4427.11 and 4526.95). Three peaks (<i>m/z</i> 3624.65, 3794.74 and 3808.88) were detected but not identified. Bottom panel: the <i>m/z</i> peak corresponding to the non-glycosylated triacylated LppX_1–29 peptide was specifically observed in the Δ<i>ppm1</i>mutant (<i>m/z</i> 3779.28, in bold). The <i>m/z</i> 3795.28 peak was not identified. • = 1 hexose (Δm = 162 Da). • = unknown modification (Δm = 262 Da). “ni” means not identified and asterisks indicate that <i>m/z</i> assignments are not very accurate. Inset: SDS PAGE of the purified LppX proteins from the wild type and the Δ<i>ppm1</i> strains.</p

<i>M. tuberculosis</i> LppX is O-glycosylated in <i>C. glutamicum.</i>

<p><b>A.</b> Localization of LppX in <i>C. glutamicum</i> wild-type strain: Membrane (M) and secreted (S) proteins were analyzed by SDS-PAGE, followed by immunoblotting using monoclonal anti-his antibodies (left). Purification of LppX: LppX was purified and analyzed by SDS PAGE before (lane 1) or after Triton X114 extraction. Proteins from both aqueous (lane 2) and detergent (lane 3) phases were precipitated and loaded on the gel (right). LC-ESI-MS/MS analysis of LppX peptides: Identified peptides generated by standard extraction procedures are shown in boldface type on the amino acid sequence of recombinant LppX. * indicates hydroxyl amino acid residues of the LppX_6–29 peptide. <b>B.</b> LC-MS analysis of LppX_6–29 glycopeptides. The deconvoluted LC-MS chromatogram is shown (21.5+1 ions corresponding to the unmodified peptide (<i>m/z</i> 2462.17), glycosylated forms with 1 to 4 hexose units (<i>m/z</i> 2624.21, 2786.26, 2948.30, 3110.35) and Δ262- glycosylated forms with 0 to 3 hexose units (<i>m/z</i> 2724.23, 2886.28, 3048.34, 3210.37) are shown. • = 1 hexose (Δm = 162). • = unknown modification (Δm = 262). <b>C.</b> Deconvoluted MS/MS spectra of unmodified and tetraglycosylated peptides. Fragmentation patterns of the triply charged ions corresponding to unmodified (left) and tetraglycosylated (right) LppX_6–29 peptides are shown. b8, y8 and y16 most intense fragment ions are annotated in both MS/MS spectra. Fragmentation pattern of the tetraglycosylated LppX_6–29 peptidereveals 4 neutral losses of 162 Da coming from the intact tetraglycosylated peptide (<i>m/z</i> 3110.38) and from the tetraglycosylated y16 fragment ion (<i>m/z</i> 2285.03). • = 1 hexose (Δm = 162 Da).</p

Cg-Ppm2 activity affects LppX glycosylation.

<p>Comparison of MALDI PMF profiles of LppX protein purified from ΔΔ<i>ppm2</i>, Δ<i>ppm2</i> (pCg-<i>ppm2</i>) and Δ<i>ppm2</i> (pCg-<i>ppm1</i>). The <i>m/z</i> 3500–4550 region of the mass spectra of LppX tryptic peptides after DDM/CHCl₃-CH₃OH treatment is shown and significant monoisotopic [M+H]⁺¹ peaks are annotated. Upper spectrum: in the the Δ<i>ppm2</i> strain, only the <i>m/z</i> peak corresponding to the non-glycosylated diacylated LppX_1–29 peptide is identified (<i>m/z</i> 3540.89, bold). Five peaks were assigned but not identified (<i>m/z</i> 3624.64, 3635.96, 3794.77, 3808.89 and 4518.28). These peaks do not match to internal tryptic LppX peptides. Middle spectrum: in the Δ<i>ppm2</i> (pCg-<i>ppm2</i>) strain, <i>m/z</i> peaks corresponding to different glycosylated forms of the triacylated LppX_1–29 peptide are observed (<i>m/z</i> 3941.15, 4041.13 and 4103.22, in bold) as well as the peak corresponding to the non-glycosylated triacylated LppX_1–29 peptide (<i>m/z</i> 3779.23, in bold). The <i>m/z</i> 3624.63 peak was detected but not identified. Bottom spectrum: in the Δ<i>ppm2</i> (pCg-<i>ppm1</i>) strain different glycosylated forms of the diacylated LppX_1–29 peptide are detected (<i>m/z</i> 3864.99, 3964,86, 4188.82 and 4288.85, in bold). • = 1 hexose (ΔΔm = 162 Da). • = unknown modification (Δm = 262 Da). “ni” means not identified and asterisks indicate that <i>m/z</i> assignments are not very accurate. Inset: SDS PAGE of the purified LppX proteins from the wild type, the Δ<i>ppm2</i> and the complemented strains.</p

Cg-Ppm2 exhibits apolipoprotein N-acyltransferase activity. A.

<p>Localization of AmyE in <i>C. glutamicum</i> Δ<i>ppm2</i> or Δ<i>ppm2</i> (Cg-<i>ppm2</i>) strains. Membrane (M) and secreted (S) proteins were analyzed by SDS-PAGE followed by immunoblotting using monoclonal anti-his antibodies. The band labeled with an asterisk corresponds to a shorter form of AmyE. <b>B.</b> MALDI PMFs of AmyE protein purified from ΔCg-<i>ppm2</i> and ΔCg-<i>ppm2</i> (Cg-<i>ppm2</i>) strains. The <i>m/z</i> 3200–4800 region of the mass spectra of AmyE tryptic peptides after DDM/CHCl₃-CH₃OH treatment is shown and significant monoisotopic [M+H]⁺¹ peaks are annotated. Upper panel: <i>m/z</i> 3858.98 (bold) corresponds to the diacylated AmyE_1–29 peptide while <i>m/z</i> 3451.65, 3547.59 and 4045.88 match to internal tryptic peptides, AmyE_334–365, AmyE_60–91 and AmyE_55–91, respectively. Bottom panel: <i>m/z</i> 4097.21 (bold) corresponds to the triacylated AmyE_1–29 peptide while <i>m/z</i> 3451.65, 3547.61 and 4045.88 match to AmyE_334–365, AmyE_60–91 and AmyE_55–91 peptides.</p

Α(1→6)-Mannosyltransferase activity in membranes prepared from , Δ, Δ pVWEx-Cg- and Δ pVWEx-Mt

<p><b>Copyright information:</b></p><p>Taken from "Identification of an α(1→6) mannopyranosyltransferase (MptA), involved in lipomanann biosynthesis, and identification of its orthologue in "</p><p></p><p>Molecular Microbiology 2007;65(6):1503-1517.</p><p>Published online Jan 2007</p><p>PMCID:PMC2157549.</p><p>© 2007 The Authors; Journal compilation © 2007 Blackwell Publishing Ltd</p> A. Biosynthetic reaction scheme of products formed in the α(1→6)-mannosyltransferase assay utilizing α--Man-(1→6)-α--Man-C and C-PP[C]M. B. α(1→6)-Mannosyltransferase activity determined using the synthetic α--Man-(1→6)-α--Man-C neoglycolipid acceptor in a cell-free assay using 1 mg of membrane protein as described previously (). The products of the assay were re-suspended in -butanol before scintillation counting. The incorporation of [C]Man was determined by subtracting counts present in control assays (incubations in the absence of acceptor), which were typically less than 100 cpm per assay. The remaining labelled material was subjected to TLC using silica gel plates (5735 silca gel 60F, Merck) developed in CHCl/CHOH/HO/NHOH (65:25:3.6:0.5, v/v/v/v) and the products visualized by phosphorimaging (Kodak K Screen). The results represent triplicate assays in two independent experiments

Lipoglycan profiles of , , Δ and Δ Lipoglycans were analysed using SDS-PAGE and visualized using a Pro-Q emerald glycoprotein stain (Invitrogen) specific for carbohydrates

<p><b>Copyright information:</b></p><p>Taken from "Identification of an α(1→6) mannopyranosyltransferase (MptA), involved in lipomanann biosynthesis, and identification of its orthologue in "</p><p></p><p>Molecular Microbiology 2007;65(6):1503-1517.</p><p>Published online Jan 2007</p><p>PMCID:PMC2157549.</p><p>© 2007 The Authors; Journal compilation © 2007 Blackwell Publishing Ltd</p> The three major bands represented by Cg-LAM, Cg-LM and Cg-t-LM are indicated. The STD lane contains CandyCane glycoprotein molecular weight standards (Invitrogen). The four major bands represent glycoproteins of 180, 82, 42 and 18 kDa respectively