Papers by Laurie Reinhardt
Biochemistry, Jan 5, 2018
There is considerable interest in how "second shell" interactions between protein side ... more There is considerable interest in how "second shell" interactions between protein side chains and metal ligands might modulate Mn(II) ion redox properties and reactivity in metalloenzymes. One such Mn-dependent enzyme is oxalate decarboxylase (OxDC), which catalyzes the disproportionation of oxalate monoanion into formate and CO2. EPR studies have shown that a non-heme, mononuclear Mn(III) ion is formed in OxDC during catalytic turnover and that removal of a hydrogen bond between one of the metal ligands (Glu101) and a conserved, second shell tryptophan residue (Trp132) gives rise to altered zero-field splitting parameters for the catalytically important Mn(II) ion. We now report heavy-atom kinetic isotope effect measurements on the W132F OxDC variant, which test the hypothesis that the Glu101/Trp132 hydrogen bond modulates the stability of the Mn(III) form of the enzyme. Our results suggest that removing the Glu101/Trp132 hydrogen bond increases the energy of the transiti...
Journal of Agricultural and Food Chemistry, 2020
The protein precipitation (PP) of bovine serum albumin (BSA), lysozyme (LYS), and alfalfa leaf pr... more The protein precipitation (PP) of bovine serum albumin (BSA), lysozyme (LYS), and alfalfa leaf protein (ALF) by four procyanidin-rich condensed tannin (CT) samples in both 2-[Nmorpholino]ethanesulfonic acid (MES) and a modified Goering-Van Soest (GVS) buffer is described. Purified CT samples examined include Vitis vinifera seed (mean degree of polymerization [mDP] 4.1, 16.5 % galloylated), Tilia sp. flowers (B-type linkages, mDP 5.9), Vaccinium macrocarpon berries (mDP 8.7, 31.7% A-type linkages) and Trifolium pratense flowers (B-type linkages, mDP 12.3) and were characterized by 2D NMR (>90% purity). In general, CTs precipitated ALF >LYS≥BSA. PP in GVS buffer was 1 to 2.25 times greater than in MES buffer (25 °C). The GVS buffer system better reflects the results/conclusions from the literature on the impact mDP, galloylation and A-type linkages have on PP. Determinations of PP using the MES buffer at 37 °C indicated that some of these differences may be attributed to the temperature GVS buffer determinations are conducted. In vitro PP studies using the GVS buffer may offer better guidance when selecting CT-containing forages and amendments for ruminant feeding studies.
Molecules (Basel, Switzerland), Jan 23, 2018
Previous studies showed that a series of purified condensed tannins (CTs) from warm-season perenn... more Previous studies showed that a series of purified condensed tannins (CTs) from warm-season perennial legumes exhibited high variability in their modulation of methane production during in vitro rumen digestion. The molecular weight differences between these CTs did not provide correlation with either the in vitro CH₄ production or the ability to precipitate bovine serum albumin. In an effort to delineate other structure-activity relationships from these methane abatement experiments, the structures of purified CTs from these legumes were assessed with a combination of methanolysis, quantitative thiolysis, ¹H-C HSQC NMR spectroscopy and ultrahigh-resolution MALDI-TOF MS. The composition of these CTs is very diverse: procyanidin/prodelphinidin (PC/PD) ratios ranged from 98/2 to 2/98; cis/trans ratios ranged from 98/2 to 34/66; mean degrees of polymerization ranged from 6 to 39; and % galloylation ranged from 0 to 75%. No strong correlation was observed between methane production and t...
Journal of Agricultural and Food Chemistry, 2017
Unambiguous investigation of condensed tannin (CT) structure−activity relationships in biological... more Unambiguous investigation of condensed tannin (CT) structure−activity relationships in biological systems requires well-characterized, high-purity CTs. Sephadex LH-20 and Toyopearl HW-50F resins were compared for separating CTs from acetone/water extracts, and column fractions analyzed for flavan-3-ol subunits, mean degree of polymerization (mDP), and purity. Toyopearl HW-50F generated fractions with higher mDP values and better separation of procyanidins (PC) and prodelphinidins (PD) but required a prepurification step, needed more time for large scale purifications, and gave poorer recoveries. Therefore, two gradient elution schemes were developed for CT purification on Sephadex LH-20 providing 146−2000 mg/fraction. Fractions were analyzed by thiolysis and NMR spectroscopy. In general, PC/PD ratios decreased and mDP increased during elution. 1 H NMR spectroscopy served as a rapid screening tool to qualitatively determine CT enrichment and carbohydrate impurities present, guiding fractionation toward repurification or 1 H− 13 C HSQC NMR spectroscopy and thiolysis. These protocols provide options for preparing highly pure CT samples.
with specic help available everywhere you see the i O symbol. The following versions of software ... more with specic help available everywhere you see the i O symbol. The following versions of software and data (see references i O) were used in the production of this report:
Biomass and Bioenergy, 2020
Traditional sugarcane breeding seeks for high sucrose content. However, with the increasing deman... more Traditional sugarcane breeding seeks for high sucrose content. However, with the increasing demand for renewable energy sources, there has been a shift in desired traits for sugarcane as an energy crop. Energy cane is a cultivar developed from Saccharum spp. with high fiber, higher biomass productivity and resilience, having great potential as a bioenergy feedstock. This work analyzed biomass content and chemical composition of two energy cane clones (VG1126 and VIGNIS 3) and one of its progenitors, S. spontaneum (VG spo), throughout distinct stages of internode development. VG1126 had a fiber and sucrose content closer to the ancestral, showing 24% more fiber than VIGNIS 3, being more suitable for industrial lignocellulose applications. Both energy canes showed an increase in lignin content, S/G ratio and pCA along internode development. Ancestral cane, however, showed similar composition across culm development, suggesting that it completes vegetative growth faster than energy cane. Although the three crops have different fiber contentconte, sucrose content, and morphology, their overall biomass composition (lignin, cell wall sugars, and hydroxycinnamates) was similar. These results represent a useful model to follow chemical cell wall composition during vegetative cell development and are expected to help breeding energy cane cultivars programs to use in biorefineries.
Biochemistry, 2016
Oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into formate and carbon dioxide ... more Oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into formate and carbon dioxide in a remarkable reaction that requires manganese and dioxygen. Previous studies have shown that replacing an active-site loop segment Ser(161)-Glu(162)-Asn(163)-Ser(164) in the N-terminal domain of OxDC with the cognate residues Asp(161)-Ala(162)-Ser-(163)-Asn(164) of an evolutionarily related, Mn-dependent oxalate oxidase gives a chimeric variant (DASN) that exhibits significantly increased oxidase activity. The mechanistic basis for this change in activity has now been investigated using membrane inlet mass spectrometry (MIMS) and isotope effect (IE) measurements. Quantitative analysis of the reaction stoichiometry as a function of oxalate concentration, as determined by MIMS, suggests that the increased oxidase activity of the DASN OxDC variant is associated with only a small fraction of the enzyme molecules in solution. In addition, IE measurements show that C-C bond cleavage in the DASN OxDC variant proceeds via the same mechanism as in the wild-type enzyme, even though the Glu(162) side chain is absent. Thus, replacement of the loop residues does not modulate the chemistry of the enzyme-bound Mn(II) ion. Taken together, these results raise the possibility that the observed oxidase activity of the DASN OxDC variant arises from an increased level of access of the solvent to the active site during catalysis, implying that the functional role of Glu(162) is to control loop conformation. A 2.6 Å resolution X-ray crystal structure of a complex between oxalate and the Co(II)-substituted ΔE162 OxDC variant, in which Glu(162) has been deleted from the active site loop, reveals the likely mode by which the substrate coordinates the catalytically active Mn ion prior to C-C bond cleavage. The "end-on" conformation of oxalate observed in the structure is consistent with the previously published V/K IE data and provides an empty coordination site for the dioxygen ligand that is thought to mediate the formation of Mn(III) for catalysis upon substrate binding.
Journal of the Chemical Society, Perkin Transactions 2, 1999
Science, 2007
Biotin-dependent multifunctional enzymes carry out metabolically important carboxyl group transfe... more Biotin-dependent multifunctional enzymes carry out metabolically important carboxyl group transfer reactions and are potential targets for the treatment of obesity and type 2 diabetes. These enzymes use a tethered biotin cofactor to carry an activated carboxyl group between distantly spaced active sites. The mechanism of this transfer has remained poorly understood. Here we report the complete structure of pyruvate carboxylase at 2.0 angstroms resolution, which shows its domain arrangement. The structure, when combined with mutagenic analysis, shows that intermediate transfer occurs between active sites on separate polypeptide chains. In addition, domain rearrangements associated with activator binding decrease the distance between active-site pairs, providing a mechanism for allosteric activation. This description provides insight into the function of biotin-dependent enzymes and presents a new paradigm for multifunctional enzyme catalysis.
Journal of the American Chemical Society, 1998
The nature of the hydrogen bonding in complexes of alkylimidazoles and substituted carboxylic aci... more The nature of the hydrogen bonding in complexes of alkylimidazoles and substituted carboxylic acids has been studied as a model of the hydrogen-bonding interaction of the proton bridging N δ1 of His 57 and the carboxyl group of Asp 102 in the active site of chymotrypsin. The interaction has been postulated to be a low barrier hydrogen bond (LBHB) in the enzyme and also in the model complexes which have a small ∆pK a . In the present study, enthalpies of complex formation, -∆H formation , between alkylimidazoles (1-methyl, 1-n-butyl-, and 1-tert-butylimidazole) and a series of carboxylic acids were determined by adiabatic solution calorimetry in chloroform. In FTIR studies presented here, the concentration of LBHB present in these complexes was determined. For complexation between dichloropropionic acid and alkylimidazoles for which the ∆pK a is small in chloroform, the -∆H formation values varied from 12 to 15 kcal/mol. Thus in enzymes, where ∆G is similar to ∆H, ∆G formation can be as high as -12 to -15 kcal/mol for LBHBs. If a weak hydrogen bond in the initial E‚substrate complex with a ∆G formation of e-5 kcal/mol is converted to a low barrier hydrogen bond in the transition state, there will be 7-10 kcal/mol of energy available to lower the activation barrier and accelerate the reaction by 5-7 orders of magnitude.
Journal of the American Chemical Society, 2003
Oxalate decarboxylase (OxDC) catalyzes a remarkable transformation in which the C-C bond in oxala... more Oxalate decarboxylase (OxDC) catalyzes a remarkable transformation in which the C-C bond in oxalate is cleaved to give carbon dioxide and formate. Like the native OxDC isolated from Aspergillus niger, the recombinant, bacterial OxDC from Bacillus subtilis contains Mn(II) in its resting state and requires catalytic dioxygen for activity. The most likely mechanism for OxDC-catalyzed C-C bond cleavage involves the participation of free radical intermediates, although this hypothesis remains to be unequivocally demonstrated. Efforts to delineate the catalytic mechanism have been placed on a firm foundation by the high-resolution crystal structure of recombinant, wild type B. subtilis OxDC (Anand et al., Biochemistry 2002, 41, 7659-7669). We now report the results of heavy-atom kinetic isotope effect measurements for the OxDC-catalyzed decarboxylation of oxalate, in what appear to be the first detailed studies of the mechanism employed by OxDC. At pH 4.2, the OxDC-catalyzed formation of formate and CO2 have normal 13 C isotope effects of 1.5% ( 0.1% and 0.5% ( 0.1%, respectively, while the 18 O isotope effect on the formation of formate is 1.1% ( 0.2% normal. Similarly at pH 5.7, the production of formate and CO2 exhibits normal 13 C isotope effects of 1.9% ( 0.1% and 0.8% ( 0.1%, respectively, and the 18 O isotope effect on the formation of formate is 1.0% ( 0.2% normal. The 18 O isotope effect on the formation of CO2, however, 0.7% ( 0.2%, is inverse at pH 5.7. These results are consistent with a multistep model in which a reversible, proton-coupled, electron transfer from bound oxalate to the Mn-enzyme gives an oxalate radical, which decarboxylates to yield a formate radical anion. Subsequent reduction and protonation of this intermediate then gives formate.
Journal of the American Chemical Society, 1999
Pressure effects on the ionization of various compounds were determined by pressure-modulated flu... more Pressure effects on the ionization of various compounds were determined by pressure-modulated fluoresence spectroscopy of buffering solutions containing nanomolar fluorescein as a pH indicator. Two highly pressure-sensitive buffers are described. The second ionization of 1,1-cyclopropane dicarboxylic acid (pK 7.5) has a ∆V ion of -29 ( 2 mL/mol, the largest known aqueous ionization volume of any Brönsted acid of pK a below 9. The third ionization of 1,4,8,12-tetraazacyclopentadecane exhibits a ∆V ion of +30 ( 2 mL/mol, the largest positive aqueous ionization volume known for any Brönsted base in water. The 1 H NMR spectrum of the tetrabutylammonium salt of 1,1-cyclopropane dicarboxylic acid monoanion in acetone-d 6 with 10% H 2 O at 25°C had a peak at 19.25 ppm, suggesting that the two carbonyl groups are tethered by a low-barrier hydrogen bond which shields electron density from the solvent and that ionization of this proton leads to a large increase in solvation of the carboxylates. The protonation of 1,4,8,12-tetraazacyclopentadecane dication causes a structural change that is accompanied by a large increase in charge density and may contribute to the large ionization volume via enhancement of electrostriction.
Journal of Physical Organic Chemistry, 2001
The vertical ionization potentials for three tetra-a-branched-alkyl-substituted alkenes (13-15) a... more The vertical ionization potentials for three tetra-a-branched-alkyl-substituted alkenes (13-15) are reported, compared with literature data for other tetralkylalkenes, and the values discussed with reference to AM1 semiempirical and B3LYP density functional calculations.
The Journal of Organic Chemistry, 2006
Multiple isotope effects were measured at the reactive center of formamide during acid-catalyzed ... more Multiple isotope effects were measured at the reactive center of formamide during acid-catalyzed hydrolysis in water at 25 degrees C. The mechanism involves a rapid pre-equilibrium protonation of the carbonyl oxygen, followed by the formation of at least one tetrahedral intermediate, which does not appreciably exchange its carbonyl oxygen with the solvent (kh/kex = 55). The pKa for formamide was determined by 15N NMR and found to be about -2.0. The formyl-hydrogen kinetic isotope effect (KIE) is indicative of a transition state that is highly tetrahedral (Dkobs = 0.79); the carbonyl-carbon KIE (13kobs = 1.031) is in agreement with this conclusion. The small leaving-nitrogen KIE (15kobs = 1.0050) is consistent with some step prior to breaking the C-N bond as rate-determining. The carbonyl-oxygen KIE (18kobs = 0.996) points to attack of water as the rate-determining step. On the basis of these results, a mechanism is proposed in which attachment of the nucleophile to a protonated formamide molecule is rate determining.
FEBS Letters, 2013
Galactokinase (GALK), a member the Leloir pathway for normal galactose metabolism, catalyzes the ... more Galactokinase (GALK), a member the Leloir pathway for normal galactose metabolism, catalyzes the conversion of α-d-galactose to galactose-1-phosphate. For this investigation, we studied the kinetic mechanism and pH profiles of the enzyme from Lactococcus lactis. Our results show that the mechanism for its reaction is sequential in both directions. Mutant proteins D183A and D183N are inactive (< 10000 fold), supporting the role of Asp183 as a catalytic base that deprotonates the C-1 hydroxyl group of galactose. The pH-kcat profile of the forward reaction has a pKa of 6.9 ± 0.2 that likely is due to Asp183. The pH-k(cat)/K(Gal) profile of the reverse reaction further substantiates this role as it is lacking a key pKa required for a direct proton transfer mechanism. The R36A and R36N mutant proteins show over 100-fold lower activity than that for the wild-type enzyme, thus suggesting that Arg36 lowers the pKa of the C-1 hydroxyl to facilitate deprotonation.
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Papers by Laurie Reinhardt