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Stephen L. Bearne - One of the best experts on this subject based on the ideXlab platform.

  • altering the y137 k164 k166 triad of Mandelate racemase and its effect on the observed pka of the bronsted base catalysts
    Archives of Biochemistry and Biophysics, 2019
    Co-Authors: Christopher M Fetter, Mitesh Nagar, Zachary A Morrison, Colin D Douglas, Stephen L. Bearne
    Abstract:

    Abstract Mandelate racemase (MR) catalyzes the interconversion of the enantiomers of Mandelate using a two-base mechanism with Lys 166 acting as the Bronsted base to abstract the α-proton from (S)-Mandelate. The resulting intermediate is subsequently re-protonated by the conjugate acid of His 297 to yield (R)-Mandelate. The roles of these amino acids are reversed when (R)-Mandelate is the substrate. The side chains of Tyr 137, Lys 164, and Lys 166 form a H-bonding network and the proximity of the two e-NH3+ groups is believed to lower the pKa of Lys 166. We used site-directed mutagenesis, kinetics, and pH-rate studies to explore the roles of Lys 164 (K164 C/M) and Tyr 137 (Y137  L/F/S/T) in catalysis. The efficiency (kcat/Km) was reduced ∼3.5 × 105-fold for K164C MR, relative to wild-type MR, indicating a major role for this residue in catalysis. The efficiency of Y137F MR, however, was reduced only 25–30-fold. pH-Rate profiles (log kcat vs. pH) revealed that substitution of Tyr 137 by Phe increased the kinetic pKa of Lys 166 from 5.88 ± 0.02 to 7.3 ± 0.2. Hence, Tyr 137 plays an important role in facilitating the reduction of the pKa of the Bronsted base Lys 166 by ∼1.4 units. Interestingly, the Phe substitution also increased the kinetic pKa of His 297 from 5.97 ± 0.04 to 7.1 ± 0.1. Thus, the Tyr 137-Lys 164-Lys 166 H-bonding network plays a broader role in modulating the pKa of catalytic residues by influencing the electrostatic character of the entire active site, not only by decreasing the observed pKa value of Lys 166, but also by decreasing the pKa of His 297 by 1.1 units.

  • catalytic properties of the metal ion variants of Mandelate racemase reveal alterations in the apparent electrophilicity of the metal cofactor
    Metallomics, 2019
    Co-Authors: Matthew Harty, Amar Nath Sharma, Stephen L. Bearne
    Abstract:

    Mandalate racemase (MR) from Pseudomonas putida requires a divalent metal cation, usually Mg2+, to catalyse the interconversion of the enantiomers of Mandelate. Although the active site Mg2+ may be replaced by Mn2+, Co2+, or Ni2+, substitution by these metal ions does not markedly (<10-fold) alter the kinetic parameters Kappm, kappcat, and (kcat/Km)app for the substrates (R)- and (S)-Mandelate, and the alternative substrate (S)-trifluorolactate. Viscosity variation experiments with Mn2+-MR showed that the metal ion plays a role in the uniform binding of the transition states for enzyme-substrate association, the chemical step, and enzyme-product dissociation. Surprisingly, the competitive inhibition constants (Ki) for inhibition of each metalloenzyme variant by benzohydroxamate did not vary significantly with the identity of the metal ion unlike the marked variation of the stability constants (K1) observed for M2+·BzH complex formation in solution. A similar trend was observed for the inhibition of the metalloenzyme variants by F-, except for Mg2+-MR, which bound F- tighter than would be predicted based on the stability constants for formation of M2+·F- complexes in solution. Thus, the enzyme modifies the enatic state of the bound metal ion cofactor so that the apparent electrophilicity of Mg2+ is enhanced, while that of Ni2+ is attenuated, resulting in a levelling effect relative to the trends observed for the free metals in solution.

  • an additional role for the bronsted acid base catalysts of Mandelate racemase in transition state stabilization
    Biochemistry, 2015
    Co-Authors: Mitesh Nagar, Stephen L. Bearne
    Abstract:

    Mandelate racemase (MR) catalyzes the interconversion of the enantiomers of Mandelate and serves as a paradigm for understanding the enzyme-catalyzed abstraction of an α-proton from a carbon acid substrate with a high pKa. The enzyme utilizes a two-base mechanism with Lys 166 and His 297 acting as Bronsted acid and base catalysts, respectively, in the R → S reaction direction. In the S → R reaction direction, their roles are reversed. Using isothermal titration calorimetry (ITC), MR is shown to bind the intermediate/transition state (TS) analogue inhibitor benzohydroxamate (BzH) in an entropy-driven process with a value of ΔCp equal to −358 ± 3 cal mol–1 K–1, consistent with an increased number of hydrophobic interactions. However, MR binds BzH with an affinity that is ∼2 orders of magnitude greater than that predicted solely on the basis of hydrophobic interactions [St. Maurice, M., and Bearne, S. L. (2004) Biochemistry 43, 2524], suggesting that additional specific interactions contribute to binding. To...

  • Inactivation of Mandelate Racemase by 3-Hydroxypyruvate Reveals a Potential Mechanistic Link between Enzyme Superfamilies.
    Biochemistry, 2015
    Co-Authors: Mitesh Nagar, Martin St. Maurice, Brittney N. Wyatt, Stephen L. Bearne
    Abstract:

    Mandelate racemase (MR), a member of the enolase superfamily, catalyzes the Mg2+-dependent interconversion of the enantiomers of Mandelate. Several α-keto acids are modest competitive inhibitors of MR [e.g., mesoxalate (Ki = 1.8 ± 0.3 mM) and 3-fluoropyruvate (Ki = 1.3 ± 0.1 mM)], but, surprisingly, 3-hydroxypyruvate (3-HP) is an irreversible, time-dependent inhibitor (kinact/KI = 83 ± 8 M–1 s–1). Protection from inactivation by the competitive inhibitor benzohydroxamate, trypsinolysis and electrospray ionization tandem mass spectrometry analyses, and X-ray crystallographic studies reveal that 3-HP undergoes Schiff-base formation with Lys 166 at the active site, followed by formation of an aldehyde/enol(ate) adduct. Such a reaction is unprecedented in the enolase superfamily and may be a relic of an activity possessed by a promiscuous progenitor enzyme. The ability of MR to form and deprotonate a Schiff-base intermediate furnishes a previously unrecognized mechanistic link to other α/β-barrel enzymes util...

  • potent inhibition of Mandelate racemase by a fluorinated substrate product analogue with a novel binding mode
    Biochemistry, 2014
    Co-Authors: Mitesh Nagar, Martin St. Maurice, A D Lietzan, Stephen L. Bearne
    Abstract:

    Mandelate racemase (MR) from Pseudomonas putida catalyzes the Mg2+-dependent 1,1-proton transfer that interconverts the enantiomers of Mandelate. Because trifluorolactate is also a substrate of MR, we anticipated that replacing the phenyl rings of the competitive, substrate-product analogue inhibitor benzilate (Ki = 0.7 mM) with trifluoromethyl groups might furnish an inhibitor. Surprisingly, the substrate-product analogue 3,3,3-trifluoro-2-hydroxy-2-(trifluoromethyl)propanoate (TFHTP) was a potent competitive inhibitor [Ki = 27 ± 4 μM; cf. Km = 1.2 mM for both (R)-Mandelate and (R)-trifluorolactate]. To understand the origins of this high binding affinity, we determined the X-ray crystal structure of the MR–TFHTP complex to 1.68 A resolution. Rather than chelating the active site Mg2+ with its glycolate moiety, like other ground state analogues, TFHTP exhibited a novel binding mode with the two trifluoromethyl groups closely packed against the 20s loop and the carboxylate bridging the two active site Bro...

Yun-chao Kan - One of the best experts on this subject based on the ideXlab platform.

  • high level and enantioselective production of l phenylglycine from racemic mandelic acid by engineered escherichia coli using response surface methodology
    Enzyme and Microbial Technology, 2020
    Co-Authors: Cun-duo Tang, Hong-ling Shi, Yuan-yuan Jia, Lun-guang Yao, Linfeng Wang, Yun-chao Kan
    Abstract:

    Abstract L-Phenylglycine (L-PHG) is a member of unnatural amino acids, and becoming more and more important as intermediate for pharmaceuticals, food additives and agrochemicals. However, the existing synthetic methods for L-PHG mainly rely on toxic cyanide chemistry and multistep processes. To provide green, safe and high enantioselective alternatives, we envisaged cascade biocatalysis for the one-pot synthesis of L-PHG from racemic mandelic acid. A engineered E. coli strain was established to co-express Mandelate racemase, D-Mandelate dehydrogenase and L-leucine dehydrogenase and catalyze a 3-step reaction in one pot, enantioselectively transforming racemic mandelic acid to give L-PHG (e.e. >99 %). After the conditions for biosynthesis of L-PHG optimized by response surface methodology, the yield and space-time yield of L-PHG can reach 87.89 % and 79.70 g·L−1·d−1, which was obviously improved. The high-yielding and enantioselective synthetic methods use cheap and green reagents, and E. coli whole-cell catalysts, thus providing green and useful alternative methods for manufacturing L-PHG.

  • One-Pot Synthesis of Phenylglyoxylic Acid from Racemic Mandelic Acids via Cascade Biocatalysis
    2019
    Co-Authors: Cun-duo Tang, Peng-ju Ding, Hong-ling Shi, Yuan-yuan Jia, Mao-zhi Zhou, Lun-guang Yao, Yun-chao Kan
    Abstract:

    Phenylglyoxylic acid (PGA) are key building blocks and widely used to synthesize pharmaceutical intermediates or food additives. However, the existing synthetic methods for PGA generally involve toxic cyanide and complex processes. To explore an alternative method for PGA biosynthesis, we envisaged cascade biocatalysis for the one-pot synthesis of PGA from racemic mandelic acid. A novel Mandelate racemase named ArMR showing higher expression level (216.9 U·mL–1 fermentation liquor) was cloned from Agrobacterium radiobacter and identified, and six recombinant Escherichia coli strains were engineered to coexpress three enzymes of Mandelate racemase, d-Mandelate dehydrogenase and l-lactate dehydrogenase, and transform racemic mandelic acid to PGA. Among them, the recombinant E. coli TCD 04, engineered to coexpress three enzymes of ArMR, LhDMDH, and LhLDH, can transform racemic mandelic acid (100 mM) to PGA with 98% conversion. Taken together, we provide a green approach for one-pot biosynthesis of PGA from racemic mandelic acid

  • biosynthesis of phenylglyoxylic acid by lhdmdh a novel d Mandelate dehydrogenase with high catalytic activity
    Journal of Agricultural and Food Chemistry, 2018
    Co-Authors: Cun-duo Tang, Peng-ju Ding, Hong-ling Shi, Lun-guang Yao, Zhujin Jiao, Fei Liu, Hongfei Shi, Yun-chao Kan
    Abstract:

    d-Mandelate dehydrogenase (DMDH) has the potential to convert d-mandelic acid to phenylglyoxylic acid (PGA), which is a key building block in the field of chemical synthesis and is widely used to synthesize pharmaceutical intermediates or food additives. A novel NAD+-dependent d-Mandelate dehydrogenase was cloned from Lactobacillus harbinensi (LhDMDH) by genome mining and expressed in Escherichia coli BL21. After being purified to homogeneity, the oxidation activity of LhDMDH toward d-mandelic acid was approximately 1200 U·mg–1, which was close to four times the activity of the probe. Meanwhile, the kcat/Km value of LhDMDH was 28.80 S–1·mM–1, which was distinctly higher than the probe. By coculturing two E. coli strains expressing LhDMDH and LcLDH, we developed a system for the efficient synthesis of PGA, achieving a 60% theoretical yield and 99% purity without adding coenzyme or cosubstrate. Our data supports the implementation of a promising strategy for the chiral resolution of racemic mandelic acid an...

  • Biosynthesis of Phenylglyoxylic Acid by LhDMDH, a Novel d‑Mandelate Dehydrogenase with High Catalytic Activity
    2018
    Co-Authors: Cun-duo Tang, Peng-ju Ding, Hong-ling Shi, Lun-guang Yao, Zhujin Jiao, Fei Liu, Hongfei Shi, Yun-chao Kan
    Abstract:

    d-Mandelate dehydrogenase (DMDH) has the potential to convert d-mandelic acid to phenylglyoxylic acid (PGA), which is a key building block in the field of chemical synthesis and is widely used to synthesize pharmaceutical intermediates or food additives. A novel NAD+-dependent d-Mandelate dehydrogenase was cloned from Lactobacillus harbinensi (LhDMDH) by genome mining and expressed in Escherichia coli BL21. After being purified to homogeneity, the oxidation activity of LhDMDH toward d-mandelic acid was approximately 1200 U·mg–1, which was close to four times the activity of the probe. Meanwhile, the kcat/Km value of LhDMDH was 28.80 S–1·mM–1, which was distinctly higher than the probe. By coculturing two E. coli strains expressing LhDMDH and LcLDH, we developed a system for the efficient synthesis of PGA, achieving a 60% theoretical yield and 99% purity without adding coenzyme or cosubstrate. Our data supports the implementation of a promising strategy for the chiral resolution of racemic mandelic acid and the biosynthesis of PGA

Roberto Fernandezlafuente - One of the best experts on this subject based on the ideXlab platform.

  • modulating the properties of the lipase from thermomyces lanuginosus immobilized on octyl agarose beads by altering the immobilization conditions
    Enzyme and Microbial Technology, 2020
    Co-Authors: Yuliya Lokha, Sara Aranapena, Nathalia Saraiva Rios, Carmen Mendezsanchez, Luciana Rocha Barros Goncalves, Fernando Lopezgallego, Roberto Fernandezlafuente
    Abstract:

    Abstract The lipase from Thermomyces lanuginosus (TLL) has been immobilized on octyl-agarose beads via interfacial activation under 16 different conditions (changing the immobilization pH, the ionic strength, the presence of additives like calcium, phosphate or glycerol) and using a low loading (1 mg/g support). Then, the properties of the different biocatalysts have been evaluated: stability at pH 7.0 and 70 °C and activity versus p-nitro phenyl propionate, triacetin and R- and S- methyl Mandelate. Results clearly indicate that the immobilization conditions determine the final enzyme properties, altering enzyme stability (by 10 folds), activity (by 8 folds using R- methyl Mandelate) and specificity (VR/VS changed from 0.7 to 2.3 using Mandelate esters). For instance, the enzymes immobilized at pH 7.0 using 5 mM buffer were the most stable preparations, while the presence of 250 mM sodium phosphate greatly decreased the final enzyme stability. The biocatalyst stability of TLL increased with increasing NaCl in the immobilization buffer at pH 5. Fluorescence studies confirmed that the conformation of the different immobilized enzymes were different, despite being a physical and reversible immobilization method. Thus, the immobilization of TLL on octyl agarose beads under different conditions produced biocatalysts with different properties, the optimal condition depends on the studied reaction and condition.

  • versatility of glutaraldehyde to immobilize lipases effect of the immobilization protocol on the properties of lipase b from candida antarctica
    Process Biochemistry, 2012
    Co-Authors: Rodrigo Torres, Oveimar Barbosa, Claudia Ortiz, Roberto Fernandezlafuente
    Abstract:

    Abstract Glutaraldehyde chemistry has been used to immobilize lipase B from Candida antarctica (CALB) under different situations. Using high ionic strength, ionic adsorption is avoided, but CALB is adsorbed on the support via interfacial activation. Using non-ionic detergents (e.g., Triton X-100), the enzyme becomes ionically adsorbed on the activated support. If detergent and salt are simultaneously present during immobilization, a covalent attachment to the support is first produced. In absence of detergent or high ionic strength, a mixture of all of the previous immobilization reasons should coexist. Thus, 5 different CALB biocatalysts were prepared following the previous described protocols, and its stability and activity, pH/activity profile and specificity versus R and S methyl Mandelate were analyzed. The existence of covalent attachment of more than 95% of the enzyme molecules was confirmed by washing the biocatalysts in salt and detergent solutions. The glutaraldehyde treatment of the enzyme adsorbed on aminated supports did not produce a significant improvement on the activity of the enzyme versus p-nitrophenylpropinate (pNPB) nor a high stabilization of the enzyme. This differed from the effects of a similar treatment of CAL adsorbed on octyl agarose. However, they were similar to the effects of this treatment on covalently immobilized CALB, suggesting that the immobilization protocol may greatly affect the final effect of a chemical modification on the enzyme properties. Dramatic changes in the enzyme features were observed comparing the different preparations, mainly in the specificity of CALB versus p-NPB and R-methyl Mandelate (from 2.5 to 20), or in the enantiospecificity versus R/S methyl Mandelate (from 1.8 to 16), confirming that these different immobilization protocols produced biocatalysts with different features. Moreover, changes in experimental conditions produced very different effects on the properties of the different CALB preparations.

Bharati Mitra - One of the best experts on this subject based on the ideXlab platform.

  • structure of an active soluble mutant of the membrane associated s Mandelate dehydrogenase
    Biochemistry, 2001
    Co-Authors: Narayanasami Sukumar, Bharati Mitra, Domenico L Gatti, F S Mathews
    Abstract:

    The structure of an active mutant of (S)-Mandelate dehydrogenase (MDH-GOX2) from Pseudomonas putida has been determined at 2.15 A resolution. The membrane-associated flavoenzyme (S)-Mandelate dehydrogenase (MDH) catalyzes the oxidation of (S)-Mandelate to give a flavin hydroquinone intermediate which is subsequently reoxidized by an organic oxidant residing in the membrane. The enzyme was rendered soluble by replacing its 39-residue membrane-binding peptide segment with a corresponding 20-residue segment from its soluble homologue, glycolate oxidase (GOX). Because of their amphipathic nature and peculiar solubilization properties, membrane proteins are notoriously difficult to crystallize, yet represent a large fraction of the proteins encoded by genomes currently being deciphered. Here we present the first report of such a structure in which an internal membrane-binding segment has been replaced, leading to successful crystallization of the fully active enzyme in the absence of detergents. This approach may have general application to other membrane-bound proteins. The overall fold of the molecule is that of a TIM barrel, and it forms a tight tetramer within the crystal lattice that has circular 4-fold symmetry. The structure of MDH-GOX2 reveals how this molecule can interact with a membrane, although it is limited by the absence of a membrane-binding segment. MDH-GOX2 and GOX adopt similar conformations, yet they retain features characteristic of membrane and globular proteins, respectively. MDH-GOX2 has a distinctly electropositive surface capable of interacting with the membrane, while the opposite surface is largely electronegative. GOX shows no such pattern. MDH appears to form a new class of monotopic integral membrane protein that interacts with the membrane through coplanar electrostatic binding surfaces and hydrophobic interactions, thus combining features of both the prostaglandin synthase/squaline-hopine cyclase and the C-2 coagulation factor domain classes of membrane proteins.

  • s Mandelate dehydrogenase from pseudomonas putida mutations of the catalytic base histidine 274 and chemical rescue of activity
    Biochemistry, 1999
    Co-Authors: Isabelle E Lehoux, Bharati Mitra
    Abstract:

    (S)-Mandelate dehydrogenase from Pseudomonas putida, an FMN-dependent alpha-hydroxy acid dehydrogenase, oxidizes (S)-Mandelate to benzoylformate. The generally accepted catalytic mechanism for this enzyme involves the formation of a carbanion intermediate. Histidine-274 has been proposed to be the active-site base that abstracts the substrate alpha-proton to generate the carbanion. Histidine-274 was altered to glycine, alanine, and asparagine. All three mutants were completely inactive. The mutants were able to form adducts with sulfite, though with much weaker affinity than the wild-type enzyme. Binding of the inhibitor, (R)-Mandelate, was not greatly affected by the mutation, unlike that of the substrate, (S)-Mandelate, indicating that H274 plays a role in substrate binding. The activity of H274G and, to a lesser extent, H274A could be partially restored by the addition of exogenous imidazoles. The maximum rescued activity for H274G with imidazole was approximately 0.1% of the wild-type value. Saturation kinetics obtained for rescued activity suggest that formation of a ternary complex of imidazole, enzyme, and substrate is required for catalysis. pH-dependence studies confirm that the free base form of imidazole is the rescue agent. An earlier study of pH profiles of the wild-type enzyme indicated that deprotonation of a residue with a pK(a) of 5.5 in the free enzyme was essential for activity (Lehoux, I. E., and Mitra, B. (1999) Biochemistry 38, 5836-5848). Data obtained in this work confirm that the pK(a) of 5.5 belongs to histidine-274.

  • mechanism of the reaction catalyzed by Mandelate racemase structure and mechanistic properties of the d270n mutant
    Biochemistry, 1995
    Co-Authors: Abraham T Kallarakal, Gregory A Petsko, John W Kozarich, Ja Gerlt, Bharati Mitra, James R Clifton, George L Kenyon
    Abstract:

    On the basis of the available high-resolution structures of Mandelate racemase (MR) from Pseudomonas putida [Landro, J. A., Gerlt, J. A., Kozarich, J. W., Koo, C. W., Shah, V. J., Kenyon, G. L., Neidhart, D. J., Fujita, J., & Petsko, G. A. (1994) Biochemistry 33, 635−643], Lys 166 and His 297 are positioned appropriately to participate in catalysis as acid/base catalysts, with Lys 166 participating as the (S)-specific acid/base catalyst and His 297 participating as the (R)-specific acid/base catalyst. The dependence of kcat on pH for the racemization of both (R)- and (S)-Mandelates suggests that the pKas of the conjugate acids of Lys 166 and His 297 are both ∼6.4 [Landro, J. A., Kallarakal, A. T., Ransom, S. C., Gerlt, J. A., Kozarich, J. W., Neidhart, D. J., & Kenyon, G. L. (1991) Biochemistry 30, 9274−9281; Kallarakal, A. T., Mitra, B., Kozarich, J. W., Gerlt, J. A., Clifton, J. R., Petsko, G. A., & Kenyon, G. L. (1995) Biochemistry 34, 2788−2797]. Both acid/base catalysts are in close proximity to and ...

  • mechanism of the reaction catalyzed by Mandelate racemase importance of electrophilic catalysis by glutamic acid 317
    Biochemistry, 1995
    Co-Authors: Bharati Mitra, Gregory A Petsko, John W Kozarich, Ja Gerlt, Abraham T Kallarakal, James G Clifton, George L Kenyon
    Abstract:

    In the high-resolution X-ray structure of Mandelate racemase (MR) with the competitive inhibitor (S)-atrolactate bound in the active site [Landro, J. A., Gerlt, J. A., Kozarich, J. W., Koo, C. W., Shah, V. J., Kenyon, G. L., Neidhart, D. J., Fujita, J., & Petsko, G. A. (1994) Biochemistry 33, 635-643], the carboxylic acid group of Glu 317 is hydrogen-bonded to the carboxylate group of the bound inhibitor. This geometry suggests that the carboxylic acid functional group of Glu 317 participates as a general acid catalyst in the concerted general acid-general base catalyzed formation of a stabilized enolic tautomer of mandelic acid as a reaction intermediate. To test this hypothesis, the E317Q mutant of MR was constructed and subjected to high-resolution X-ray structural analysis in the presence of (S)-atrolactate. No conformational alterations were observed to accompany the E317Q substitution at 2.1 A resolution. The values for kcat were reduced 4.5 x 10(3)-fold for (R)-Mandelate and 2.9 x 10(4)-fold for (S)-Mandelate; the values for kcat/Km were reduced 3 x 10(4)-fold. The substrate and solvent deuterium isotope effects measured for both wild-type MR and the E317Q mutant are not multiplicative when deuteriated substrate is studied in D2O, which suggests that the reactions catalyzed by both enzymes are stepwise and involve the formation of stabilized enolic intermediates. In contrast to wild-type MR, E317Q does not catalyze detectable elimination of bromide ion from either enantiomer of p-(bromomethyl)Mandelate. However, E317Q is irreversibly inactivated by racemic alpha-phenylglycidate at a rate comparable to that measured for wild-type MR. Taken together, these mechanistic properties confirm the importance of Glu 317 as a general acid catalyst in the reaction catalyzed by wild-type MR. The kcat for wild-type MR and the reduction in kcat observed for E317O are discussed in terms of the analysis recently described by Gerlt and Gassman for understanding the rates and mechanisms of enzyme-catalyzed proton abstraction reactions from carbon acids [Gerlt, J. A., & Gassman, P. G. (1993) J. Am. Chem. Soc. 115, 11552-11568; Gerlt, J. A., & Gassman, P. G. (1993) Biochemistry 32, 11943-11952].

  • a novel structural basis for membrane association of a protein construction of a chimeric soluble mutant of s Mandelate dehydrogenase from pseudomonas putida
    Biochemistry, 1993
    Co-Authors: Bharati Mitra, Ja Gerlt, George L Kenyon, Carolyn W Koo, Patricia C Babbitt, Diane Joseph, Gregory A Petsko
    Abstract:

    The (S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida (ATCC 12633) is the only membrane-associated member of a homologous family of FMN-dependent, alpha-hydroxy acid dehydrogenases/oxidases that includes the structurally characterized glycolate oxidase from spinach (GOX). We have correlated the membrane association of MDH to a polypeptide segment in the interior of the primary sequence. This has been accomplished by construction of a chimeric enzyme in which the putative membrane-binding segment in MDH has been deleted and replaced with the corresponding segment from the soluble GOX. The resulting chimera, MDH-GOX, is soluble and retains partial catalytic activity (approximately 1%) using (S)-Mandelate as substrate. In contrast, the activities of both the membrane-associated wild-type MDH and the soluble MDH-GOX are nearly the same when (S)-phenyllactate is used as substrate. To the best of our knowledge, this is the first example of a membrane-associated protein in which an internal polypeptide segment anchors the protein to the membrane.

Jun Huang - One of the best experts on this subject based on the ideXlab platform.

  • catalytic performance of bronsted and lewis acid sites in phenylglyoxal conversion on flame derived silica zirconia
    Chemcatchem, 2014
    Co-Authors: Zichun Wang, Yijiao Jiang, Alfons Baiker, Michael Hunger, Jun Huang
    Abstract:

    Flame-derived silica–zirconia has been used for the promising one-step catalytic conversion of phenylglyoxal (PG) to Mandelates, important intermediates in pharmacy and fine chemistry. In the literature it was proposed that Lewis acid sites (LAS) on solid acids promote Mandelate production, whereas Bronsted acid sites (BAS) only generate acetal byproducts during PG conversion. Herein it is shown that ZrO2, which contains only LAS and no BAS, exhibits a very low activity for PG conversion to afford a yield of Mandelate of only 8 % after 6 h. The activity of this ZrO2 catalyst was confirmed by the condensation of acetone, in which its LAS could activate the carbonyl groups immediately for the fast condensation of all acetone molecules. The introduction of BAS by the admixing of silica precursor to the feed in the flame-spray pyrolysis enhanced the yield of Mandelate up to 52–67 %. This indicates that BAS are mainly responsible for the PG conversion to Mandelate on silica–zirconia catalysts and that their LAS are nearly inactive for this reaction.

  • efficient acid catalyzed conversion of phenylglyoxal to Mandelates on flame derived silica alumina
    ACS Catalysis, 2013
    Co-Authors: Zichun Wang, Yijiao Jiang, Alfons Baiker, Jun Huang
    Abstract:

    Amorphous, nonporous silica/alumina (SA) made by flame-spray pyrolysis (FSP) efficiently catalyzes the direct conversion of phenylglyoxal (PG) to alkyl Mandelates. The SAs exhibited a turnover frequency more than an order of magnitude higher than dealuminated zeolite Y, which hitherto has been considered as the most active solid acid for this reaction. The free diffusion of PG to surface acid sites and rapid removal of Mandelate products are proposed to be at the origin of the superior performance of SAs. The recyclability of the catalyst was tested in five repetitive runs and showed no significant loss of catalyst performance.