Absence Of Substrate Research Articles

Reactivating chaperones for coenzyme B12-dependent diol and glycerol dehydratases and ethanolamine ammonia-lyase.

Adenosylcobalamin (AdoCbl) or coenzyme B12-dependent enzymes tend to undergo mechanism-based inactivation during catalysis or inactivation in the absence of substrate. Such inactivation may be inevitable because they use a highly reactive radical for catalysis, and side reactions of radical intermediates result in the damage of the coenzyme. How do living organisms address such inactivation when enzymes are inactivated by undesirable side reactions? We discovered reactivating factors for radical B12 eliminases. They function as releasing factors for damaged cofactor(s) from enzymes and thus mediate their exchange for intact AdoCbl. Since multiple turnovers and chaperone functions were demonstrated, they were renamed "reactivases" or "reactivating chaperones." They play an essential role in coenzyme recycling as part of the activity-maintaining systems for B12 enzymes. In this chapter, we describe our investigations on reactivating chaperones, including their discovery, gene cloning, preparation, characterization, activity assays, and mechanistic studies, that have been conducted using a wide range of biochemical and structural methods that we have developed.

Capture of activated dioxygen intermediates at the copper-active site of a lytic polysaccharide monooxygenase.

Metalloproteins perform a diverse array of redox-related reactions facilitated by the increased chemical functionality afforded by their metallocofactors. Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-dependent enzymes that are responsible for the breakdown of recalcitrant polysaccharides via oxidative cleavage at the glycosidic bond. The activated copper-oxygen intermediates and their mechanism of formation remains to be established. Neutron protein crystallography which permits direct visualization of protonation states was used to investigate the initial steps of oxygen activation directly following active site copper reduction in Neurospora crassa LPMO9D. Herein, we cryo-trap an activated dioxygen intermediate in a mixture of superoxo and hydroperoxo states, and we identify the conserved second coordination shell residue His157 as the proton donor. Density functional theory calculations indicate that both superoxo and hydroperoxo active site states are stable. The hydroperoxo formed is potentially an early LPMO catalytic reaction intermediate or the first step in the mechanism of hydrogen peroxide formation in the absence of substrate. We observe that the N-terminal amino group of the copper coordinating His1 remains doubly protonated directly following molecular oxygen reduction by copper. Aided by molecular dynamics and mining minima free energy calculations we establish that the conserved second-shell His161 in MtPMO3* displays conformational flexibility in solution and that this flexibility is also observed, though to a lesser extent, in His157 of NcLPMO9D. The imidazolate form of His157 observed in our structure following oxygen intermediate protonation can be attributed to abolished His157 flexibility due steric hindrance in the crystal as well as the solvent-occluded active site environment due to crystal packing. A neutron crystal structure of NcLPMO9D at low pH further supports occlusion of the active site since His157 remains singly protonated even at acidic conditions.

Reverse Ordered Sequential Mechanism for Lactoperoxidase with Inhibition by Hydrogen Peroxide.

Lactoperoxidase (LPO, FeIII in its resting state in the absence of substrates)—an enzyme secreted from human mammary, salivary, and other mucosal glands—catalyzes the oxidation of thiocyanate (SCN−) by hydrogen peroxide (H2O2) to produce hypothiocyanite (OSCN−), which functions as an antimicrobial agent. The accepted catalytic mechanism, called the halogen cycle, comprises a two-electron oxidation of LPO by H2O2 to produce oxoiron(IV) radicals, followed by O-atom transfer to SCN−. However, the mechanism does not explain biphasic kinetics and inhibition by H2O2 at low concentration of reducing substrate, conditions that may be biologically relevant. We propose an ordered sequential mechanism in which the order of substrate binding is reversed, first SCN− and then H2O2. The sequence of substrate binding that is described by the halogen cycle mechanism is actually inhibitory.

Purification and kinetic properties of a novel β-amylase from Penicillum citrinum AS-9

β-amylase-rich preparation produced by the soil fungal strain Penicillium citrinum AS-9 was completely purified within the succeeding steps; ultrafiltration, acetone fractionation and gel filtration on Sephadex G-150 column. Acetone fractionation recovered the highest β-amylase activity and the 85% acetone fraction exhibited 3.5-fold activity that of the crude enzyme. The column affected 8.6-fold purification for β-amylase, which produced maltose as the major product of starch hydrolysis. The pure enzyme showed a Km value of 17.5 mM and Vmax of 17.5 Umg-1 protein, applying Woolf plot and exhibited its maximum velocity at pH 7.1 and 50°C. In absence of substrate, thermal treatments of the enzyme solution at pH 5.2 and 5.5 had the most adverse effects on the enzyme activity. CaCl2 (1mM) activated the enzyme, while each of Ca2+, Fe3+, cysteine, cystine, I2 and p-chloromercuribenzoate (PCMB) had different inhibitory effects. Maltose as the enzyme product at final concentration of 80 mM strongly inhibited the enzyme.

Binding interactions in a kinase active site modulate background ATP hydrolysis

Kinases play central roles in many cellular processes, transferring the terminal phosphate groups of nucleoside triphosphates (NTPs) onto substrates. In the absence of substrates, kinases can also hydrolyse NTPs producing NDPs and inorganic phosphate. Hydrolysis is usually much less efficient than the native phosphoryl transfer reaction. This may be related to the fact that NTP hydrolysis is metabolically unfavorable as it unproductively consumes the cell's energy stores. It has been suggested that substrate interactions could drive changes in NTP binding pocket, activating catalysis only when substrates are present. Structural data show substrate-induced conformational rearrangements, however there is a lack of corresponding functional information. To better understand this phenomenon, we developed a suite of isothermal titration calorimetry (ITC) kinetics methods to characterize ATP hydrolysis by the antibiotic resistance enzyme aminoglycoside-3′-phosphotransferase-IIIa (APH(3′)-IIIa). We measured Km, kcat, and product inhibition constants and single-turnover kinetics in the presence and absence of non-substrate aminoglycosides (nsAmgs) that are structurally similar to the native substrates. We found that the presence of an nsAmg increased the chemical step of cleaving the ATP γ-phosphate by at least 10- to 20-fold under single-turnover conditions, supporting the existence of interactions that link substrate binding to substantially enhanced catalytic rates. Our detailed kinetic data on the association and dissociation rates of nsAmgs and ADP shed light on the biophysical processes underlying the enzyme's Theorell-Chance reaction mechanism. Furthermore, they provide clues on how to design small-molecule effectors that could trigger efficient ATP hydrolysis and generate selective pressure against bacteria harboring the APH(3′)-IIIa.

Human flavin-containing monooxygenase 1 and its long-sought hydroperoxyflavin intermediate

Out of the five isoforms of human flavin-containing monooxygenase (hFMO), FMO1 and FMO3 are the most relevant to Phase I drug metabolism. They are involved in the oxygenation of xenobiotics including drugs and pesticides using NADPH and FAD as cofactors. Majority of the characterization of these enzymes has involved hFMO3, where intermediates of its catalytic cycle have been described. On the other hand, research efforts have so far failed in capturing the same key intermediate that is responsible for the monooxygenation activity of hFMO1. In this work we demonstrate spectrophotometrically the formation of a highly stable C4a-hydroperoxyflavin intermediate of hFMO1 upon reduction by NADPH and in the presence of O2. The measured half-life of this flavin intermediate revealed it to be stable and not fully re-oxidized even after 30 min at 15 °C in the absence of substrate, the highest stability ever observed for a human FMO. In addition, the uncoupling reactions of hFMO1 show that this enzyme is <1% uncoupled in the presence of substrate, forming small amounts of H2O2 with no observable superoxide as confirmed by EPR spin trapping experiments. This behaviour is different from hFMO3, that is shown to form both H2O2 and superoxide anion radical as a result of ∼50% uncoupling. These data are consistent with the higher stability of the hFMO1 intermediate in comparison to hFMO3. Taken together, these data demonstrate the different behaviours of these two closely related enzymes with consequences for drug metabolism as well as possible toxicity due to reactive oxygen species.

Structures of ABCG2 under turnover conditions reveal a key step in the drug transport mechanism

ABCG2 is a multidrug transporter that affects drug pharmacokinetics and contributes to multidrug resistance of cancer cells. In previously reported structures, the reaction cycle was halted by the absence of substrates or ATP, mutation of catalytic residues, or the presence of small-molecule inhibitors or inhibitory antibodies. Here we present cryo-EM structures of ABCG2 under turnover conditions containing either the endogenous substrate estrone-3-sulfate or the exogenous substrate topotecan. We find two distinct conformational states in which both the transport substrates and ATP are bound. Whereas the state turnover-1 features more widely separated NBDs and an accessible substrate cavity between the TMDs, turnover-2 features semi-closed NBDs and an almost fully occluded substrate cavity. Substrate size appears to control which turnover state is mainly populated. The conformational changes between turnover-1 and turnover-2 states reveal how ATP binding is linked to the closing of the cytoplasmic side of the TMDs. The transition from turnover-1 to turnover-2 is the likely bottleneck or rate-limiting step of the reaction cycle, where the discrimination of substrates and inhibitors occurs.

A thermophilic, pH-tolerant, and highly active GH10 xylanase from Aspergillus fumigatus boosted pre-treated sugarcane bagasse saccharification by cellulases

We have heterogously expressed a recombinant GH10 xylanase from Aspergillus fumigatus Af293 (Af-XYLA) in Pichia pastoris X-33 and investigated its biochemical and kinetic properties and its synergy with cellulases during lignocellulose sacchararification. Af-XYLA showed optimal activity at 80 °C and pH 5–7, and it withstood a temperature of 50 °C and pH 4–8 for 72 h with about 50 % and 80 % of its maximal activity, respectively, in the absence of substrate. Kinetic assays revealed that it presented the highest Vmax (35,756 U mg−1 at 50 °C and 88,082 U mg−1 at 80 °C) and kcat/KM (15,456 mL mg−1 s−1 at 50 °C and 15,081 mL mg−1 s−1 at 80 °C) constants among fungal GH10 xylanases. Af-XYLA was xylose- and glucose-tolerant and displayed >70 % activity at 10 % (v/v) ethanol. Few additives affected Af-XYLA negatively, and the surfactants Tween 20, Triton X-100, and SLS and the reducing agents DTT and β-mercaptoethanol improved enzyme performance by 28 %, 11 %, 8%, 44 %, and 32 %, respectively. We applied a 3-factor Central Composite Rotational Design (CCRD) to optimize important parameters for biomass saccharification and thus maximize the reducing sugars yields obtained from sugarcane exploded bagasse (SEB) and sugarcane delignified bagasse (SDB) hydrolyses. We carried out synergism assays under the previously determined optimal concentrations of Celluclast 1.5®L (1.8 FPU g−1), Af-XYLA (3.75 μg g−1), and Tween 20 (0.75 % v/v). Af-XYLA boosted SEB and SDB hydrolyses by cellulases by 155 % and 277 %, respectively, after 48 h. On the basis of the unique properties of Af-XYLA, it is a robust enzyme for application in biorefineries.

Structural and Biochemical Characterization of a Dye-Decolorizing Peroxidase from Dictyostelium discoideum.

A novel cytoplasmic dye-decolorizing peroxidase from Dictyostelium discoideum was investigated that oxidizes anthraquinone dyes, lignin model compounds, and general peroxidase substrates such as ABTS efficiently. Unlike related enzymes, an aspartate residue replaces the first glycine of the conserved GXXDG motif in Dictyostelium DyPA. In solution, Dictyostelium DyPA exists as a stable dimer with the side chain of Asp146 contributing to the stabilization of the dimer interface by extending the hydrogen bond network connecting two monomers. To gain mechanistic insights, we solved the Dictyostelium DyPA structures in the absence of substrate as well as in the presence of potassium cyanide and veratryl alcohol to 1.7, 1.85, and 1.6 Å resolution, respectively. The active site of Dictyostelium DyPA has a hexa-coordinated heme iron with a histidine residue at the proximal axial position and either an activated oxygen or CN− molecule at the distal axial position. Asp149 is in an optimal conformation to accept a proton from H2O2 during the formation of compound I. Two potential distal solvent channels and a conserved shallow pocket leading to the heme molecule were found in Dictyostelium DyPA. Further, we identified two substrate-binding pockets per monomer in Dictyostelium DyPA at the dimer interface. Long-range electron transfer pathways associated with a hydrogen-bonding network that connects the substrate-binding sites with the heme moiety are described.

Detection of an enzyme isomechanism by means of the kinetics of covalent inhibition

Turnover of substrates by many enzymes involves free enzyme forms that differ from the stable form of the enzyme in the absence of substrate. These enzyme species, known as isoforms, have, in general, different physical and chemical properties than the native enzymes. They usually occur only in small concentrations under steady state turnover conditions and thus are difficult to detect. We show in this paper that in one particular case of an enzyme (a class C β-lactamase) with specific substrates (cephalosporins) the presence of an enzyme isoform (E′) can be detected by means of its different reactivity than the native enzyme (E) with a class of covalent inhibitors (phosphonate monoesters). Generation of E′ from E arises either directly from substrate turnover or by way of a branched path from an acyl-enzyme intermediate. The relatively slow spontaneous restoration of E from E′ is accelerated by certain small molecules in solution, for example cyclic amines such as imidazole and salts such as sodium chloride. Solvent deuterium kinetic isotope effects and the effect of methanol on cephalosporin turnover showed that for both E and E′, kcat is limited by deacylation of an acyl-enzyme intermediate rather than by enzyme isomerization.

Investigation of the pH-dependence of the oxidation of FAD in VcCRY-1, a member of the cryptochrome-DASH family.

Vibrio cholerae cryptochrome-1 (VcCRY-1) is a member of the cryptochrome DASH family. The flavoprotein appears to use blue light both for repair of cyclobutane pyrimidine dimers (CPDs) on DNA and signal transduction. Earlier, we found that it was almost impossible to oxidize the FADH· state upon binding to a CPD, and, in the absence of substrate, the rate of FADH· oxidation was much larger at high pH (Gindt et al. in Biochemistry 54:2802-2805, 2015). Here, we present the pH-dependence of the oxidation of FADH· by ferricyanide, which revealed a switch between slow and fast oxidation with a pKa ≈ 7.0. Stopped-flow mixing was used to measure the oxidation of FADH- to FADH· at pH 6.7 and 7.5. Substrate binding was required to slow down this oxidation such that it could be measured with stopped flow, but there was only a small effect of pH. In addition, resonance Raman measurements of FADH· in VcCRY-1 at pH 6.5 and 7.5 were performed to probe for structural changes near the FAD cofactor related to the observed changes in rate of FADH· oxidation. Only substrate binding seemed to induce a change near the FAD cofactor that may relate to the change in oxidation kinetics. The pH-effect on the FADH· oxidation rate, which is rate-limited by the proton acceptor, does not seem to be due to a protein structural change near the FAD cofactor. Instead, a conserved glutamate in CRY-DASH may control the deprotonation of FADH· and give rise to the pH-effect.

Can use of substrates affect water quality in aquatic organism culture?

The choice of suitable substrates has received special attention in aquaculture and fishkeeping ventures. The aim of this study was to evaluate the influence of different substrates on water quality and the development of lambari fish (Astyanax bimaculatus) and freshwater shrimp (Macrobrachium potiuna). Two experiments were performed sequentially, one with fish and the other with shrimp. In both experiments, three different substrates were evaluated (shell gravel, river gravel, and dolomite), and the control was without any substrate. The water parameters evaluated were as follows: dissolved oxygen, saturation, oxide-reduction potential, temperature, pH, alkalinity, conductivity, ammonia, nitrite, nitrate, and phosphorus. It was observed that both dolomite and shell gravel interfered with the parameters that are related to the acid-base balance of the water and the availability of calcium and magnesium. The productive performance of lambari fish was not affected considering the different substrates evaluated; however, the productive performance of freshwater shrimp was lower in the absence of substrates in relation to the other treatments.

Reaction coupling in ADH1A alcohol dehydrogenase enzyme by exciplex formation with adenosine diphosphate moderated by low-energy electronic excited states.

Two commonly accepted theories about enzymes were revisited. The first states that adenosine triphosphate (ATP)-stored energy is only released when the substrate is in place, because the substrate changes the enzyme structure when it is bound to the enzyme. In fact, as demonstrated and discussed presently, no structural changes are required, and ATP-stored energy is released when it can be used. The second states that ATP-released energy moves along the enzyme molecule in the form of molecular vibrations (Davydov's vibrational solitons). In fact, as reported presently, energy released upon ATP hydrolysis moves in the form of excited-state electrons (excitons), with no molecular vibrations involved. The relevant experimental evidence was obtained for the human ADH1A alcohol dehydrogenase enzyme. Spontaneous ATP hydrolysis in the absence of substrate was apparently prevented by electronically excited enzyme + adenosine diphosphate (ADP) + inorganic phosphate (P) complex (exciplex) formed upon ATP hydrolysis. This exciplex kept ADP + P bound and in place for the inverse reaction, until the excess energy was dissipated in the enzyme-catalyzed reaction or by energy transfer to a suitable acceptor. Additionally, and contrary to textbooks, ADH1A has required ATP, working orders of magnitude faster in its presence.

Role of non-covalent interactions at the oxidation of 2,5-di-Me-pyrazine-di-N-oxide at glassy carbon, single-walled and multi-walled carbon nanotube paper electrodes

Recently in our work it was shown that the catalytic efficiency of organic compound oxidation in the presence of electrochemically generated radical cations of aromatic di-N-oxides was increased several times using single-walled (SWCNT) or multi-walled (MWCNT) carbon nanotube paper electrodes instead of glassy carbon (GC) electrode. It was found that, in the absence of substrate, the oxidation currents of di-N-oxides at SWCNT or MWCNT paper electrodes, in contrast to the GC electrode, exceeded the oxidation current of the ferrocene (Fc) reference several times. In this work, the study of 2,5-di-Me-pyrazine-di-N-oxide (Pyr1) and Fc oxidation in 0.1 M Bu4NClO4 solutions in acetonitrile (MeCN) at GC, SWCNT and MWCNT paper electrodes was performed by methods of cyclic voltammetry, electron paramagnetic resonance (EPR) electrolysis, and differential capacitance. Quantum chemical modeling of adsorption of Pyr1, Fc, and MeСN on CNT surface was carried out using a cluster model describing the surface of conducting and non-conducting carbon nanotubes. The adsorption energies, equilibrium distances for molecular location and orientation on CNT surface were obtained. The observed effects were explained on the basis of quantum chemical modeling of the non-covalent interaction of the components of the studied system with CNT surface.

Oligomeric assembly regulating mitochondrial HtrA2 function as examined by methyl-TROSY NMR

Human High temperature requirement A2 (HtrA2) is a mitochondrial protease chaperone that plays an important role in cellular proteostasis and in regulating cell-signaling events, with aberrant HtrA2 function leading to neurodegeneration and parkinsonian phenotypes. Structural studies of the enzyme have established a trimeric architecture, comprising three identical protomers in which the active sites of each protease domain are sequestered to form a catalytically inactive complex. The mechanism by which enzyme function is regulated is not well understood. Using methyl transverse relaxation optimized spectroscopy (TROSY)-based solution NMR in concert with biochemical assays, a functional HtrA2 oligomerization/binding cycle has been established. In the absence of substrates, HtrA2 exchanges between a heretofore unobserved hexameric conformation and the canonical trimeric structure, with the hexamer showing much weaker affinity toward substrates. Both structures are substrate inaccessible, explaining their low basal activity in the absence of the binding of activator peptide. The binding of the activator peptide to each of the protomers of the trimer occurs with positive cooperativity and induces intrasubunit domain reorientations to expose the catalytic center, leading to increased proteolytic activity. Our data paint a picture of HtrA2 as a finely tuned, stress-protective enzyme whose activity can be modulated both by oligomerization and domain reorientation, with basal levels of catalysis kept low to avoid proteolysis of nontarget proteins.

Perspective of Using Gluconacetobacter sucrofermentas VKPM B-11267 in Biofuel Cells

The bioelectrochemical and spectral properties of immobilized Gluconacetobacter sucrofermentas VKPM B-11267 bacteria were studied in the presence and absence of multiwalled carbon nanotubes (MWCNTs). The obtained characteristics were compared with the characteristics of Gluconobacter oxydans, which are phylogenetically close to them and are widely used in bioelectrochemistry. It was shown that modification of the bioelectrode with carbon nanotubes leads to a significant increase in the current level (by 2.5–3 times), as well as to a decrease in the total resistance both in the absence of substrates and in their presence. The potential use of immobilized G. sucrofermentas cells as part of a microbial fuel cell (MFC) was considered. The specific electrical power of an MFC based on immobilized G. sucrofermentas cells was lower than that of an MFC based on G. oxydans cells. Nevertheless, the results obtained indicate that G. sucrofermentas VKPM B-11267 cells can serve as a biocatalyst in MFCs.

Characterization of the sorbitol dehydrogenase SmoS from Sinorhizobium meliloti 1021.

Sinorhizobium meliloti 1021 is a Gram-negative alphaproteobacterium with a robust capacity for carbohydrate metabolism. The enzymes that facilitate these reactions assist in the survival of the bacterium across a range of environmental niches, and they may also be suitable for use in industrial processes. SmoS is a dehydrogenase that catalyzes the oxidation of the commonly occurring sugar alcohols sorbitol and galactitol to fructose and tagatose, respectively, using NAD+ as a cofactor. The main objective of this study was to evaluate SmoS using biochemical techniques. The nucleotide sequence was codon-optimized for heterologous expression in Escherichia coli BL21 (DE3) Gold cells and the protein was subsequently overexpressed and purified. Size-exclusion chromatography and X-ray diffraction experiments suggest that SmoS is a tetramer. SmoS was crystallized, and crystals obtained in the absence of substrate diffracted to 2.1 Å resolution and those of a complex with sorbitol diffracted to 2.0 Å resolution. SmoS was characterized kinetically and shown to have a preference for sorbitol despite having a higher affinity for galactitol. Computational ligand-docking experiments suggest that tagatose binds the protein in a more energetically favourable complex than fructose, which is retained in the active site over a longer time frame following oxidation and reduces the rate of the reaction. These results supplement the inventory of biomolecules with potential for industrial applications and enhance the understanding of metabolism in the model organism S. meliloti.

Conformational Transition Pathway of GkPOT

GkPOT, a Proton-Coupled coupled Oligopeptide Transporter transporter (POTs) from Geobacillus Kaustophilus, is among the symporter members of the Major Facilitator Superfamily (MFS) of transporters. Functional and structural analysis of various POTs suggest that the uptake of small peptides and peptide- like molecules to the cell across the cell membrane, via the alternating access mechanism, utilizes an inward directed proton electrochemical gradient the source of energy for their active symport function. The transition between the inward-facing (IF) and outward-facing (OF) conformations is important in understanding the structural relationship between large-scale protein conformational changes and substrate translocation that has often times remained elusive due to the short time scales of molecular dynamics simulations (MD) limitations of computational and experimental effort techniques. Using an extensive set of enhanced sampling techniques, we have characterized the conformational transition pathway of GkPOT. We have aimed at describing the large-scale conformational changes between the IF and OF states while in the presence and absence of substrate (dialanine peptide). Nonequlibrium pulling simulations were initiated from the known inward facing IF conformation and steered the system toward the unknown outward facing OF conformation using orientation based collective variables defined on different transmembrane helices. The transition pathways are far from equilibrium and are smoothed with string method with swarms of trajectories (SMwST), thus generating microseconds of biased and unbiased simlulations. Free energy profiles of the conformational transition pathways were obtained from a novel maximum-likelihood estimation method. Our results indicate that the presence of ligand significantly changes the conformational landscape with respect to the apo system which contains a greater degree of conformational variability.

Ultrafast dynamics of fully reduced flavin in catalytic structures of thymidylate synthase ThyX.

Thymidylate is a vital DNA precursor synthesized by thymidylate synthases. ThyX is a flavin-dependent thymidylate synthase found in several human pathogens and absent in humans, which makes it a potential target for antimicrobial drugs. This enzyme methylates the 2'-deoxyuridine 5'-monophosphate (dUMP) to 2'-deoxythymidine 5'-monophosphate (dTMP) using a reduced flavin adenine dinucleotide (FADH-) as prosthetic group and (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2THF) as a methylene donor. Recently, it was shown that ThyX-catalyzed reaction is a complex process wherein FADH- promotes both methylene transfer and reduction of the transferred methylene into a methyl group. Here, we studied the dynamic and photophysics of FADH- bound to ThyX, in several substrate-binding states (no substrate, in the presence of dUMP or folate or both) by femtosecond transient absorption spectroscopy. This methodology provides valuable information about the ground-state configuration of the isoalloxazine moiety of FADH- and the rigidity of its local environment, through spectra shape and excited-state lifetime parameters. In the absence of substrate, the environment of FADH- in ThyX is only mildly more constrained than that of free FADH- in solution. The addition of dUMP however narrows the distribution of ground-state configurations and increases the constraints on the butterfly bending motion in the excited state. Folate binding results in the selection of new ground-state configurations, presumably located at a greater distance from the conical intersection where excited-state decay occurs. When both substrates are present, the ground-state configuration appears on the contrary rather limited to a geometry close to the conical intersection, which explains the relatively fast excited-state decay (100 ps on the average), even if the environment of the isoalloxazine is densely packed. Hence, although the environment of the flavin is dramatically constrained, FADH- retains a dynamic necessary to shuttle carbon from folate to dUMP. Our study demonstrates the high sensitivity of FADH- photophysics to the constraints exerted by its immediate surroundings.

Hydrolytic enzymes and biodeterioration of groundnut seeds

Some common seed borne fungal isolates of Groundnut variety TAG-24 and SB-XI were screened for their ability to produce the hydrolytic enzymes. For this the test seed borne fungal isolates of Groundnut were grown on substrate and nonsubstrate glucose nitrate media for seven days at room temperature. After seven days of incubation the culture filtrates were collected separately and considered them as the crude enzyme preparation. The crude enzyme preparations were assayed by different methods under different conditions. Both varieties of Groundnut Aspergillus terreus and Sclerotium rolfsii were found to be highly amlolytic. All the test isolates of the Groundnut variety TAG-24 and SB-XI produced protease in both the media. Maximum protease production was found by Aspergillus flavus followed by Fusarium oxysporum. All the test isolates produced cellulase in both the media. Maximum cellulase production was found in Sclerotium rolfsii followed by Aspergillus niger and Aspergillus terreus. Among the seven test isolates Aspergillus flavus, Aspergillus terreus, Macrophomina phaseolina could not produce pectinase in the absence of substrate but the production was observed in the presence of substrate