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At least 235 records · Page 13
All sourcesNASA NTRSDOE OSTI
DOE OSTI2019

Structural basis for the diversity of the mechanism of nucleotide hydrolysis by the aminoglycoside-2"-phosphotransferases

Aminoglycoside phosphotransferases (APHs) are one of three families of aminoglycoside-modifying enzymes that confer high-level resistance to the aminoglycoside antibiotics via enzymatic modification. This has now rendered many clinically important drugs almost obsolete. The APHs specifically phosphorylate hydroxyl groups on the aminoglycosides using a nucleotide triphosphate as the phosphate donor. The APH(2′′) family comprises four distinct members, isolated primarily from Enterococcus sp., which vary in their substrate specificities and also in their preference for the phosphate donor (ATP or GTP). In this work, the structure of the ternary complex of APH(2′′)-IIIa with GDP and kanamycin was solved at 1.34 Å resolution and was compared with substrate-bound structures of APH(2′′)-Ia, APH(2′′)-IIa and APH(2′′)-IVa. In contrast to the case for APH(2′′)-Ia, where it was proposed that the enzyme-mediated hydrolysis of GTP is regulated by conformational changes in its N-terminal domain upon GTP binding, APH(2′′)-IIa, APH(2′′)-IIIa and APH(2′′)-IVa show no such regulatory mechanism, primarily owing to structural differences in the N-terminal domains of these enzymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Thermobifida fusca Cel6B moves bidirectionally while processively degrading cellulose

Abstract Background Cellulose, an abundant biopolymer, has great potential to be utilized as a renewable fuel feedstock through its enzymatic degradation into soluble sugars followed by sugar fermentation into liquid biofuels. However, crystalline cellulose is highly resistant to hydrolysis, thus industrial-scale production of cellulosic biofuels has been cost-prohibitive to date. Mechanistic studies of enzymes that break down cellulose, called cellulases, are necessary to improve and adapt such biocatalysts for implementation in biofuel production processes. Thermobifida fusca Cel6B ( Tf Cel6B) is a promising candidate for industrial use due to its thermostability and insensitivity to pH changes. However, mechanistic studies probing Tf Cel6B hydrolytic activity have been limited to ensemble-scale measurements. Results We utilized optical tweezers to perform single-molecule, nanometer-scale measurements of enzyme displacement during cellulose hydrolysis by Tf Cel6B. Records featured forward motility on the order of 0.17 nm s −1 interrupted by backward motions and long pauses. Processive run lengths were on the order of 5 nm in both forward and backward directions. Motility records also showed rapid bidirectional displacements greater than 5 nm. Single-enzyme velocity and bulk ensemble activity were assayed on multiple crystalline cellulose allomorphs revealing that the degree of crystallinity and hydrogen bonding have disparate effects on the single-molecule level compared to the bulk scale. Additionally, we isolated and monitored the catalytic domain of Tf Cel6B and observed a reduction in velocity compared to the full-length enzyme that includes the carbohydrate-binding module. Applied force has little impact on enzyme velocity yet it readily facilitates dissociation from cellulose. Preliminary measurements at elevated temperatures indicated enzyme velocity strongly increases with temperature. Conclusions The unexpected motility patterns of Tf Cel6B are likely due to previously unknown mechanisms of processive cellulase motility implicating irregularities in cellulose substrate ultrastructure. While Tf Cel6B is processive, it has low motility at room temperature. Factors that most dramatically impact enzyme velocity are temperature and the presence of its native carbohydrate-binding module and linker. In contrast, substrate ultrastructure and applied force did not greatly impact velocity. These findings motivate further study of Tf Cel6B for its engineering and potential implementation in industrial processes.

Johnson, Madeline M. (ORCID:000000029518293X)↗
DOE OSTI2021

High-Biomass Sorghums as a Feedstock for Renewable Fuels and Chemicals

High-biomass sorghums are intended for use in biorefineries that convert vegetative biomass into renewable fuels and chemicals. The majority of plant biomass consists of cell walls, a complex matrix of cellulose, hemicellulosic polysaccharides and lignin. In the biorefinery, the biomass is ground and then subjected to a thermo-chemical pretreatment (elevated temperature, high pressure, chemical catalysts) that disrupts the cell wall matrix and solubilizes some of the cell wall polymers, followed by enzymatic saccharification of the cellulose, which produces D-glucose. The glucose and sometimes also the xylose derived from the hydrolysis of the hemicellulosic polysaccharides, are subsequently converted to fuels or other useful chemicals by microbial biocatalysts. The genetic improvement of high-biomass sorghums has as its ultimate goals to maximize the yield of fermentable sugars on a per-hectare basis, to minimize the inputs of fertilizer, irrigation, fungicides and pesticides and to reduce the environmental footprint. Breeding strategies thus need to target biomass yield, biomass composition, maturity, pest and disease resistance and nutrient use efficiency. Furthermore, this chapter reviews the genetic basis of these traits and their potential application in breeding programs.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Enzyme Discovery in Anaerobic Fungi (Neocallimastigomycetes) Enables Lignocellulosic Biorefinery Innovation

Lignocellulosic biorefineries require innovative solutions to realize their full potential, and the discovery of novel lignocellulose-active enzymes could improve biorefinery deconstruction processes. Enzymatic deconstruction of plant cell walls is challenging, as noncarbohydrate linkages in hemicellulosic sidechains and lignin protect labile carbohydrates from hydrolysis.

Microbiology↗
DOE OSTI2026

Taste Perception of Branched Maltooligosaccharides

Oral processing of starch generates linear and branched maltooligosaccharides (MOS). This study investigates the impact of branching and the degree of polymerization (DP) on MOS taste detection. Three branched MOS samples (A−C) were enzymatically prepared from gelatinized waxy corn starch and characterized using reducing-end assays, MALDI-TOF MS, β-amylase hydrolysis, and GC-MS linkage analysis. Samples A, B, and C had average DPs of 6, 10, and 18, respectively, with α-1,6 branch points comprising 9−13% of glycosidic bonds; linear MOS were negligible. In triangle tests, participants evaluated MOS samples and a maltose control at 75 mM, with and without lactisole, a sweet receptor inhibitor. All samples were detectable without lactisole (d′ = 1.81−2.9; p < 0.05), but not with lactisole (d′ = 0−0.61; p > 0.05). These findings demonstrate that α-1,6 branching enables MOS detection via the sweet receptor, not the putative “starchy” receptor, underscoring the importance of glycosidic structure in carbohydrate taste perception.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS2022

CryoEM Structures of the Nitrogenase Complex During Catalytic Turnover

The enzyme nitrogenase couples adenosine triphosphate (ATP) hydrolysis to the multi-electron reduction of atmospheric dinitrogen into ammonia. Despite extensive research, the mechanistic details of ATP-dependent energy transduction and dinitrogen reduction by nitrogenase are not well understood, requiring new strategies to monitor its structural dynamics during catalytic action. Here we report the cryogenic electron microscopic interrogation of the nitrogenase complex under enzymatic turnover conditions, which has enabled the structural characterization of the nitrogenase reaction intermediates at high resolution for the first time. Our structures show that asymmetry governs all aspects of nitrogenase mechanism including ATP hydrolysis, protein-protein interactions, and catalysis. Furthermore, they reveal several previously unobserved, mechanistically relevant conformational changes near the catalytic iron-molybdenum cofactor that are correlated with the nucleotide-hydrolysis state of the enzyme.

Hannah L. Rutledge↗
DOE OSTI2021

Saccharide analysis of onion outer epidermal walls

Abstract Background Epidermal cell walls have special structural and biological roles in the life of the plant. Typically they are multi-ply structures encrusted with waxes and cutin which protect the plant from dehydration and pathogen attack. These characteristics may also reduce chemical and enzymatic deconstruction of the wall for sugar analysis and conversion to biofuels. We have assessed the saccharide composition of the outer epidermal wall of onion scales with different analytical methods. This wall is a particularly useful model for cell wall imaging and mechanics. Results Epidermal walls were depolymerized by acidic methanolysis combined with 2M trifluoracetic acid hydrolysis and the resultant sugars were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Total sugar yields based on wall dry weight were low (53%). Removal of waxes with chloroform increased the sugar yields to 73% and enzymatic digestion did not improve these yields. Analysis by gas chromatography/mass spectrometry (GC/MS) of per- O -trimethylsilyl (TMS) derivatives of the sugar methyl glycosides produced by acidic methanolysis gave a high yield for galacturonic acid (GalA) but glucose (Glc) was severely reduced. In a complementary fashion, GC/MS analysis of methyl alditols produced by permethylation gave substantial yields for glucose and other neutral sugars, but GalA was severely reduced. Analysis of the walls by 13 C solid-state NMR confirmed and extended these results and revealed 15% lipid content after chloroform extraction (potentially cutin and unextractable waxes). Conclusions Although exact values vary with the analytical method, our best estimate is that polysaccharide in the outer epidermal wall of onion scales is comprised of homogalacturonan (~ 50%), cellulose (~ 20%), galactan (~ 10%), xyloglucan (~ 10%) and smaller amounts of other polysaccharides. Low yields of specific monosaccharides by some methods may be exaggerated in epidermal walls impregnated with waxes and cutin and call for cautious interpretation of the results.

09 BIOMASS FUELS↗
NASA NTRS2004

Enzymatic Properties of an Alkaline and Chelator Resistant alpha-amylase from the Alkaliphilic Bacillus sp. Isolate L1711

An alkaliphilic amylase producing bacterium, Bacillus sp. strain L 711, was selected among 13 soda lakes isolates. When grown at pH 10.5 and 37 C, strain L711 produced multiple forms of amylases in the culture broth. One of these, BAA, was purified from the culture supernatant by QAE column chromatography and preparative native gel electrophoresis. The molecular weight of BAA was determined to be 51 kDa by denaturing gel electrophoresis. The pH optima for activity below and above 40 C were 9.5 - 10.0 and 7.0 - 7.5 respectively. BAA was stable in the pH range 6-11 and was completely inactivated at 55 C. The thermostability was not increased in the presence of Ca(2+). The enzyme was strongly inhibited by Ca(2+), Zn(2+), Mg(2+), Mn(2+), Ba(2+) and Cu(2+), whereas the presence of Na(+), Co(2+) and EDTA (10 mM) enhanced enzymatic activity. The K(sub m), and specific activity of BAA on soluble starch were 1.9 mg/ml and 18.5 U/mg respectively. The main end products of hydrolysis were maltotetraose, maltose and glucose.

Bernhardsdotter, Eva C. M. J.↗
NASA NTRS2004

Enzymatic Properties of an Alkaline and Chelator Resistant Proportional to alpha-Amylase from the Alkaliphilic Bacillus sp. Isolate L1711

An alkaliphilic amylase producing bacterium, Bacillus sp. strain L1711, was selected among 13 soda lakes isolates. When grown at pH 10.5 and 370 C, strain L1711 produced multiple forms of amylases in the culture broth. One of these, BAA, was purified from the culture supernatant by QAE column chromatography and preparative native gel electrophoresis. The molecular weight of BAA was determined to be 51 kDa by denaturing gel electrophoresis. The pH optima for activity below and above 40 C were 9.5-10.0 and 7.0-7.5 respectively. BAA was stable in the pH range 6-11 and was completely inactivated at 55?C. The thermostability was not increased in the presence of Ca(2+). The enzyme was strongly inhibited by Ca(2+), Zn(2+), Mg(2+), Mn(2+), Ba(2+) and Cu(2+), whereas the presence of Na(+), Co2+ and EDTA (10 mM) enhanced enzymatic activity. The K(sub m) and specific activity of BAA on soluble starch were 1.9 mg/ml and 18.5 U/mg respectively. The main end products of hydrolysis were maltotetraose, maltose and glucose .

Bernhardsdotter, Eva C. M. J.↗
DOE OSTI2025

Engineering PHL7 for Improved Poly(Ethylene Terephthalate) Depolymerization via Rational Design and Directed Evolution

Enzymatic depolymerization of poly(ethylene terephthalate) (PET) has emerged as a promising approach for polyester recycling, and, to date, many natural and engineered PET hydrolase enzymes have been reported. For industrial use, PET hydrolases must achieve high depolymerization extent and exhibit excellent thermostability. Here, we engineered a natural PET hydrolase, Polyester Hydrolase Leipzig #7 (PHL7), through rational design and directed evolution using a high-throughput screening platform. Four new enzymes were engineered with enhanced properties compared with the parent enzyme, wild-type PHL7 (PHL7-WT), and other benchmark PET hydrolases, under the tested conditions. In bioreactors, the exemplary engineered enzyme, PHL7-Jemez, exhibited improved ability to depolymerize amorphous PET film compared with PHL7-WT at 2.9% and 20% substrate loadings, with 37% and 270% higher hydrolysis, respectively, after 48 h. This study develops several state-of-the-art PET hydrolases and demonstrates a directed evolution platform to engineer high-performance enzymes, which can accelerate enzyme discovery toward improved biocatalytic recycling.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Nitrogenase Bioelectrocatalysis: ATP-Independent Ammonia Production Using a Redox Polymer/MoFe Protein System

Nitrogenase is the only biological catalyst that is understood to be able to convert nitrogen gas to ammonia. In microorganisms, the MoFe catalytic protein of nitrogenase is reduced by a transient Fe protein binding and separate hydrolysis of ATP. Yet, the requirement of 16 ATP molecules by the Fe protein for the 8 electron transfer is an energy-intense caveat to the enzymatic synthesis of NH 3 and is challenging from an electrochemical perspective. Thus, we report the redox polymer-based ATP-free mediated electron-transfer system of MoFe nitrogenase using cobaltocene-functionalized poly(allylamine) (Cc-PAA), which is able to reduce the MoFe nitrogenase directly with a low redox potential of -0.58 V vs SHE. An efficient immobilization of MoFe nitrogenase via Cc-PAA allowed for the bioelectrocatalytic reduction of N 3 – , NO 2 – , and N 2 to NH 3 . Bulk bioelectrosynthetic experiments produced 7 ± 2 and 30 ± 5 nmol of NH 3 from NO 2 – and N 3 – reduction for 30 min, respectively. In addition, biosynthetic N 2 reduction to NH 3 was confirmed by 15 N 2 labeling experiments with NMR analysis. This mediated electron-transfer approach of the immobilized nitrogenase using the Cc-PAA redox polymer provides a valuable technological basis for scale-up and industrial uses in the future of bioelectrosynthesis.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗
DOE OSTI2020

Generalized enzymatic mechanism of catalysis by tetrameric L -asparaginases from mesophilic bacteria

The mechanism of catalysis by the L -glutaminase-asparaginase from Pseudomonas 7A (PGA) was investigated using structural, mass spectrometry, and kinetic data. We had previously proposed mechanism of hydrolysis of L -Asn by the type II L-asparaginase from E. coli (EcAII), but that work was limited to just one enzyme. Based on results presented in this report, we postulate that all homotetrameric L -asparaginases from mesophilic bacteria utilize a common ping-pong mechanism of catalysis consisting of two subsequent nucleophilic substitutions. Several new structures of non-covalent complexes of PGA with different substrates, as well as structures of covalent acyl-enzyme intermediates of PGA with canonical substrates ( L -Asp and L -Glu) and an opportunistic ligand, a citrate anion, were determined. The results of kinetic experiments monitored by high-resolution LC/MS, when combined with new structural data, clearly show that the reaction catalyzed by L -glutaminase-asparaginases proceeds through formation of a covalent intermediate, as observed previously for EcAII. Additionally, by showing that the same mechanism applies to L -Asn and L -Gln, we postulate that it is common for all these structurally related enzymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

MgADP Promotes Myosin Head Movement toward Actin at Low [Ca 2+ ] to Increase Force Production and Ca 2+ -Sensitivity of Contraction in Permeabilized Porcine Myocardial Strips

Myosin cross-bridges dissociate from actin following Mg 2+ -adenosine triphosphate (MgATP) binding. Myosin hydrolyses MgATP into inorganic phosphate (P i ) and Mg 2+ -adenosine diphosphate (ADP), and release of these hydrolysis products drives chemo-mechanical energy transitions within the cross-bridge cycle to power muscle contraction. Some forms of heart disease are associated with metabolic or enzymatic dysregulation of the MgATP-MgADP nucleotide pool, resulting in elevated cytosolic [MgADP] and impaired muscle relaxation. We investigated the mechanical and structural effects of increasing [MgADP] in permeabilized myocardial strips from porcine left ventricle samples. Sarcomere length was set to 2.0 µm at 28 °C, and all solutions contained 3% dextran T-500 to compress myofilament lattice spacing to near-physiological values. Under relaxing low [Ca 2+ ] conditions (pCa 8.0, where pCa = -log 10 [Ca 2+ ]), tension increased as [MgADP] increased from 0-5 mM. Complementary small-angle X-ray diffraction measurements show that the equatorial intensity ratio, I 1,1 /I 1,0 , also increased as [MgADP] increased from 0 to 5 mM, indicating myosin head movement away from the thick-filament backbone towards the thin-filament. Ca 2+ -activated force-pCa measurements show that Ca 2+ -sensitivity of contraction increased with 5 mM MgADP, compared to 0 mM MgADP. These data show that MgADP augments tension at low [Ca 2+ ] and Ca 2+ -sensitivity of contraction, suggesting that MgADP destabilizes the quasi-helically ordered myosin OFF state, thereby shifting the cross-bridge population towards the disordered myosin ON state. Together, these results indicate that MgADP enhances the probability of cross-bridge binding to actin due to enhancement of both thick and thin filament-based activation mechanisms.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Production and Evaluation of Fluorophore-Doped Polymer Substrates to Screen for Plastic-Degrading Enzymes

Fast and sensitive analytical methods are the key to efficient screening of plastic-degrading enzymes. Here, we present a streamlined and affordable approach to assess the enzymatic deconstruction of insoluble synthetic polymers by blending them with a fluorescent dye, rhodamine 6G, and we evaluate this screening method using poly(ethylene terephthalate) (PET) as a model material. Our results indicate that enzymatic depolymerization of the rhodamine-doped PET can be observed in a high-throughput fashion by following release of the fluorophore. The fluorescence data obtained during the hydrolysis of rhodamine-doped PET by 14 PET hydrolases, produced with a robotic platform, correlated with the quantitative chromatographic analysis of PET degradation products. Remarkably, the use of the rhodamine-loaded PET substrate resulted in negligibly low background signals even when detecting PETase activity in crude cell lysates, suggesting suitability for screening of a wide variety of samples. Encouraged by these results, we next produced a selection of polyethylene- and nylon-based materials loaded with rhodamine 6G. While rapid leaching of fluorophore observed with nylon substrates limits the utility of the method for detecting nylonase activity, the rhodamine-loaded polyethylene showed promising performance in passive diffusion tests, indicating that this latter substrate may be used to screen for polyolefin-degrading enzymes.

09 BIOMASS FUELS↗
DOE OSTI2022

Beneficial effect of surfactant in adsorption/desorption of lignocellulose-degrading enzymes on/from lignin with different structure

Nonionic surfactant Tween 80 is effective in alleviating the adsorption of lignocellulose-degrading enzymes onto lignin. However, the impacts of lignin structural characteristics on this mitigation effect of Tween 80 were still not clear. Herein, effects of Tween 80 on cellulase and xylanase adsorption on different enzymatic residual lignins (ERLs) and poplar samples were investigated. Besides, effects of Tween 80 on desorption of the two enzyme preparations from different ERLs and poplar samples were also evaluated. By adding Tween 80, the inhibition of ERLs to cellulase hydrolysis was offset and the positive role of Tween 80 in Avicel hydrolysis with presence of ERLs was more effective. Binding strength of cellulase with ERLs was alleviated from 236.0 – 410.9 to 43.9 – 116.6 mL/g after adding 0.5 mg/mL Tween 80. When ERLs was more hydrophobic and had more condensed phenolic OH, the alleviation effect of Tween 80 on cellulase adsorption on ERLs was intensified but its positive effect on enhancing cellulase desorption was weakened. Tween 80 was effective in increasing xylanase activity and preventing xylanase deactivation. Further, with the increase of lignin content and phenolic OH in pretreated poplar, the effect of Tween 80 in alleviating xylanase adsorption on poplar samples was intensified but the effect in enhancing xylanase desorption from poplar samples was weakened. The results provided distinct findings to understand the interaction between Tween 80 and cellulase/xylanase in enzymatic saccharification of lignocelluloses.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1993

Is ligation the only solution to the pyrophosphate problem?

Pyrophosphate linkages are easily formed during the nonenzymatic oligomerization of activated nucleotides. They often form `caps' which terminate an oligonucleotide with a 5'-5' pyrophosphate. Owing to their structural resemblance to the intermediates in enzymatic ligation reactions, it has been suggested that pyrophosphate caps might have been capable of acting as activating groups in chain elongation processes. We argue that an alternative possibility would have been the specific hydrolysis of pyrophosphates.

Visscher, J.↗
NASA NTRS2005

Chemical evolution of RNA under hydrothermal conditions and the role of thermal copolymers of amino acids for the prebiotic degradation and formation of RNA

The roles of thermal copolymers of amino acids (TCAA) were studied for the prebiotic degradation of RNA. A weak catalytic ability of TCAA consisted of Glu, L-Ala, L-Val, L-Glu, L-Asp, and optionally L-His was detected for the cleavage of the ribose phosphodiester bond of a tetranucleotide (5'-dCrCdGdG) in aqueous solution at 80 degees C. The rate constants of the disappearance of 5'-dCrCdGdG were determined in aqueous solutions using different pH buffer and TCAA. The degradation rates were enhanced 1.3-3.0 times in the presence of TCAA at pH 7.5 and 8.0 at 80 degrees C, while the hydrolysis of oligoguanylate (oligo(G)) was accelerated about 1.6 times at pH 8.0. A weak inhibitory activity for the cleavage of oligo(G) was detected in the presence of 0.055 M TCAA-Std. On the other hand, our recent study on the influences of TCAA for the template-directed reaction of oligo(G) on a polycytidylic acid template showed that TCAA has an acceleration activity for the degradation of the activated nucleotide monomer and an acceleration activity for the formation of G5' ppG capped oligo(G). This series of studies suggest that efficient and selective catalytic or inhibitory activities for either the degradation or formation of RNA under hydrothermal conditions could have hardly emerged from the simple thermal condensation products of amino acids. A scenario is going to be deduced on the chemical evolution of enzymatic activities and RNA molecules concerning hydrothermal earth conditions. c2005 COSPAR. Published by Elsevier Ltd. All rights reserved.

Amino Acids/chemistry↗