2 Agenda Presentasi Tata nama enzim Kofaktor Mekanisme katalisis Manfaat enzimStruktur enzimTahap isolasi dan pemurnian enzimAplikasi Industri
3 IUBMB Enzyme Nomenclature EC 18.104.22.168 1 kelompok oxidoreductase, kelompok sub class 7Acting on other nitrogenous compounds as donorsKelompok subsubclass1With NAD or NADP as acceptorSumber :Springer Handbook of Enzymes
4 Kofaktor Nitrate Reductase (NR) FAD: Protein involved in flavin adenine dinucleotide synthesis or protein which contains at least one FAD as prosthetic group/cofactor (flavoprotein) such as many oxidation-reduction enzymes. FAD is an electron carrier molecule that functions as a hydrogen acceptor. The generic term "flavin" derives from the Latin word flavius ("yellow") because of the brilliant yellow color they exhibit as solids and in neutral aqueous solutions.Flavoprotein: Enzymes which contain one or more flavin nucleotides (FAD or FMN) as redox cofactors. Flavoproteins are involved, for example, in the oxidative degradation of pyruvate, fatty acids and amino acids, and in the process of electron transport.Heme: Protein containing at least one heme, an iron atom coordinated to a protoporphyrin IX. In myoglobin and hemoglobin, one of the coordination positions of iron is occupied by oxygen or other ligands, such as carbon monoxide. Hemes are also found in cytochromes of the electron-transport chain where they bind electrons, in reducing peroxides (catalases and peroxidases), and act as terminal components in multienzyme systems involved in hydroxylation. Cytochrome c is the only common heme protein in which the heme is covalently bound.Iron : Protein which binds at least one iron atom, or protein whose function is iron-dependent. Iron is a metal, chemical symbol Fe.Metal-binding: Protein which binds metals.Molybdenum: Protein which binds molybdenum (or molybdopterin) or protein involved in the transport of molybdenum, a metallic element, chemical symbol Mo. It plays an essential role in the active site of all eukaryotic Mo-containing enzymes. In plants, Mo enzymes are important for nitrate assimilation, phytohormone synthesis, and purine catabolism. Mo is often coordinated to the sulfur atoms of a pterin derivative (molybdopterin [MPT]), thereby forming the active Molybdenum cofactor (Moco), which is highly conserved in eukaryotes, eubacteria, and archaebacteria.NAD : Enzymes which use NAD(H) as an electron acceptor or as a cofactor. Nicotinamide adenine dinucleotide, an important redox coenzyme that participates in a variety of enzymatic reactions in which it serves as an electron carrier by being alternately oxidized (NAD+) and reduced (NADH). NAD also functions as an ADP-ribose donor in ADP-ribosylation reactions.
5 Mekanisme Katalisis NR The nitrate molecule binds to the active site with the Mo ion in the +6 oxidation state. Electron transfer to the active site occurs only in the proton-electron transfer stage, where the Mo(V) species plays an important role in catalysis. The presence of the sulfur atom in the molybdenum coordination sphere creates a pseudo-dithiolene ligand that protects it from any direct attack from the solvent. Upon the nitrate binding there is a conformational rearrangement of this ring that allows the direct contact of the nitrate with Mo(VI) ion. This rearrangement is stabilized by the conserved methionines Met141 and Met308. The reduction of nitrate into nitrite occurs only in the second step of the mechanism where the two dimethyl-dithiolene ligands have a key role in spreading the excess of negative charge near the Mo atom to make it available for the chemical reaction. The reaction involves the oxidation of the sulfur atoms and not of the molybdenum as previously suggested. The mechanism involves a molybdenum and sulfur-based redox chemistry instead of the currently accepted redox chemistry based only on the Mo ion. The second part of the mechanism involves two protonation steps that are promoted by the presence of Mo(V) species. Mo(VI) intermediates might also be present in this stage depending on the availability of protons and electrons. Once the water molecule is generated only the Mo(VI) species allow water molecule dissociation, and, the concomitant enzymatic turnover.Sumber: Fischer, et. al The Plant Cell.
6 Cleland Plot NRSumber: Howard, et. al Journal Biological Chemistry
7 Asimilasi NitrogenNITRATE assimilation is one of the two major biological processes by which inorganic nitrogen is converted to ammonia and thence to organic nitrogen. Photosynthetic organisms, with the possible exception of some blue-green algae and plants that have a symbiotic association with rogen-fixing bacteria, derive most of their nitrogen from nitrate1. The rate-controlling and regulated step in the process of nitrate assimilation seems to be the conversion of nitrate to nitrite, catalysed by the enzyme nitrate reductase2. There is a good correlation between the activity of this enzyme and the yield of grain protein in several cereal crops3–5. Nitrate arising from chemical fertilisers and industrial wastes is also a major factor contributing to the growth of algae and other microorganisms, leading to the eutrophication of lakes and streams. Thus, the control of nitrate assimilation can be important from the standpoint of both agricultural productivity and water resource management.
8 Stuktur 3D NRSumber: Fischer, et. al The Plant Cell
9 Sisi Aktif Enzim dan Muatan Permukaan NR Sumber: Fischer, et. al The Plant Cell
10 Arabidopsis thalianaTanaman berbunga kecil yang hidup di dataran eropa, asia, barat laut afrikaTumbuh di musim semiMempunyai siklus hidup yang relatif singkatPopuler sebagai model organisme untuk memahami biologi molekul banyak tanaman
11 Produksi Rekombinan NR NR diekspresikanDiekspresikan di methylaptrophic yeast Pickia pastoris strain GS115 dari Arabdopsisi NIA NR cDNA pAtc-4Pertumbuhan kulturDi kultur 500mL yang mengandung UPD media (1% yeast ekstrak, 2%peptone, dan 2% Glc, a11 w/v)Induksi ekspresi NRSel log phase dicuci dan diresuspensi ke dalam 1L media MM yang dimodifikasi (2,8% [w/v yeast basa nitrogen dengan ammonium sulfate, 100mM potassium phosphate, pH 6, 4x 10-5 % biotin, 0,2 mM Na-Molibdat, 1% metanolSumber: Wenpei Su, et. al Plan Physiology.
12 Produksi Rekombinan NR (Lanjt) Kultur selDitambahkan methanol 1% (v/v) dan Na-Mo (0,2 mM) setiap 24 jamSetelah 72 jam periode induksi cel dikumpulkan, disentrifuga dan diresuspensi kembali,DiresuspensiExtraction buffer mengandung 50mM sodium phospate , pH 7,3, 1 mM EDTA, dan 5% (v/v) gliserol.Sel dipecahkan dengan Bead Beater menggunakan 0,5-pm glass beads selama 5 menitEkstrak kasarDisentrifuga pada g selama 20 menit pada 4oCDiuji aktifitas ekstrak kasar dan dimurnikanRecombinant Arabidopsis NADH:NR Productionand PurificationNR was expressed in the methylotrophic yeast Pickiapastoris strain GS115 from the Arabidopsis NIA2 NR cDNApAtc-46 (Crawford et al., 1988) under the control of the P.pastoris alcohol oxidase 1 promoter as previously described(Su et al., 1996). P. pastoris was initially grown in 500-mLcultures containing YPD media (1% yeast extract, 2% peptone,and 2% Glc, a11 w/v) to an A,, of 10. The log-phasecells were washed and then resuspended to an A600 of 5 in1 L of modified MM media (2.8% [w /VI yeast nitrogen basewith ammonium sulfate, 100 mM potassium phosphate, pH6.0, 4 X 10p5% biotin, 0.2 mM sodium molybdate, and 1%methanol [v/v]) to induce NR expression. Methanol (l'h[v /VI) and sodium molybdate (0.2 mM) were added to theculture every 24 h. After a 72-h induction period the cellswere collected by centrifugation, washed, and then resuspendedin extraction buffer containing 50 mM sodiumphosphate, pH 7.3, 1 mM EDTA, and 5% (v/v) glycerol toan A600 of 50 to 100. The cells were broken with a BeadBeater (Biospec Products, Bartlesville, OK) using 0.5-pmglass beads by processing for 5 min using 30-s burstsfollowed by 1-min cooling periods. The crude extract wasthen centrifuged at 15,0008 for 20 min at 4°C.NR assays of crude extracts and purified samples wereperformed as previously described (Campbell andSmarrelli, 1978; Redinbaugh and Campbell, 1985).Sumber: Wenpei Su, et. al Plan Physiology.
13 Permunian NR Sumber: Wenpei Su, et. al. 1997. Plan Physiology. Ekstrak kasarPengendapan 30-45% amonium sulfatPeletdilarutkan padan 50mM Natrium fosfat, ph 7,3, dan 1mM EDTA (bufferA) dialirkan ke Blue-SepharoseBlue Sepharose yang berikatan dengan NR dikumpulkan dengan filtrasi vakum yang dicuci dengan kolom 1,5 cmKolom kromatografiDielusi dengan 0,1 mM NADH dalam buffer AOne unitof NR activity is defined as 1 pmol of nitrite produced/min. For the purification of wild-type NR, crude cell extractswere subjected to a 30 to 45% ammonium sulfateprecipitation. The resulting pellet was dissolved in 50 mMsodium phosphate, pH 7.3, and 1 mM EDTA (buffer A) andbatch-bound to Blue-Sepharose (Campbell and Smarrelli,1978). The Blue-Sepharose with bound NR was collected byvacuum filtration, thoroughly washed with buffer A, andpacked in a 1.5-cm column. The column was eluted with 0.1mM NADH in buffer A. Fractions containing NR activitywere pooled, buffer exchanged, and concentrated with 25mM Mops (ultrapure grade, Calbiochem), pH 7.0, using aCentriprep 30 concentrator (Amicon, Beverly, MA). Next,the partially purified sample was applied to a 1.5-mL columnof 5'-AMP Sepharose (Pharmacia).Sumber: Wenpei Su, et. al Plan Physiology.
14 Pemurnian NR (Lanjt) Sumber: Wenpei Su, et. al. 1997. Plan Physiology. Fraksi NRDipekatkan dengan 25mM Mops (ultrapure grade, Calbiochem), pH 7,0 menggunakan Centriprep 30 concetratorSampe setengah murniDialirkan ke 1,5 mL kolum 5’-AMP sepharoseKolum dicuci dengan buffer B ( 50mM Mops, pH 7, dan 1 mM EDTA.Dielusi dengan 0,1 mM NADH dalam Buffer B.Yang mengandung aktifitas 2 unit/mL disatukan dan dikonsetrasikan dengan pertukaran buffer 25 mM Mops, pH 7,0The column waswashed with 5 column volumes of 50 mM Mops, pH 7.0,and 1 mM EDTA (buffer B). NR was then eluted with 0.1mM NADH in buffer B. Fractions containing more than 2units/mL of NR activity were pooled and concentrated,with the buffer exchanged to 25 mM Mops, pH 7.0.Sumber: Wenpei Su, et. al Plan Physiology.
15 Pengujian Kinetik NRSampel NR murniEksperimen dilakukan pada 25oC dalam 1 mL buffer yang mengandung 50 mM Mops, pH 7,0 dengan pengukuran A340 dengan kuvet 1 cm dan UV /Vis SpektrofotometryKonsterasi SubtratDivariasikan dari 0,5-20 μM untuk NADH dan 2,5-60 μM untuk KNO3AnalisisKecepatan awal reaksi dianalisis dengan Enzpack softwareSpektrum dari oksidasi sampel dan NADH(red) ditentukan pada 25oC menggunakan UV/Vis dioda array spectofotometerNR samples purified as described above were used. Kineticexperiments were performed at 25'C in 1 mL of buffercontaining 50 mM Mops, pH 7.0, by following the decreasein A,,, using a 1-cm cuvette and a UV/Vis spectrophotometer(model no. 1201, Shimadzu, Kyoto, Japan). The concentrationof the substrates varied from 0.5 to 20 p~ forNADH (Sigma) and 2.5 to 60 p~ for potassium nitrate. Theresults of three initial velocity experiments were analyzedusing Enzpack software (Elsevier Biosoft, Cambridge, UK).Spectra of oxidized and NADH-reduced samples were determinedat 25°C using a UV/Vis diode array spectrophotometer(model no. 8453, Hewlett-Packard).Sumber: Wenpei Su, et. al Plan Physiology.
16 Aplikasi NR di Industri NR memiliki peran penting dalam bioteknologi lingkungan.Digunakan sebagai metode pengujian komersial kandungan nitrat dalam air (detektor elektronik berbasiskan enzim yang akan memonitoring secara kontinu kandungan nitrate dalam air)Pengujian reduks nitrate
17 ReferensiWenpei Su, Jeffrey A. Mertens, Kengo Kanamaru, Wilbur H. Campbell, and Nigel M. Crawford Analysis of Wild-Type and Mutant Plant Nitrate Reductase Expressed in the Methylotrophic Yeast Picha pastoris’. Plant Physiology.William D. Howard4 and Larry P. Solomonsonf Kinetic Mechanism of assimilatory NADH: Nitrate Reductase from Chlorella. Journal of Biological ChemistryLawrie Skipper, Wilbur H. Campbell, Jeffrey A. Mertens, and David J. Lowe Pre-steady-state Kinetic Analysis of Recombinant Arabidopsis NADH:Nitrate Reductase. JBC Papers.Katrin Fischer, Guillaume G. Barbier, Hans-Juergen Hecht, Ralf R. Mendel, Wilbur H. Campbell, and Guenter Schwarza Structural Basis of Eukaryotic Nitrate Reduction: Crystal Structures of the Nitrate Reductase Active Site. The Plant Cell.Post Translational Modification (PTM)their sequences can differ from the mere translation of their corresponding genesDisulfide bond: Protein which is modified by the formation of a bond between the thiol groups of two peptidyl-cysteine residues. The process of chemical oxidation that forms interchain disulfide bonds can produce stable, covalently linked protein dimers, multimers or complexes, whereas intrachain disulfide bonds can contribute to protein folding and stability. Depending on the protein environment, some disulfide bonds are more labile, forming transient redox-active disulfide bonds that are alternately reduced and oxidized in the course of an enzymatic reaction.Phospoprotein: Protein which is posttranslationally modified by the attachment of either a single phosphate group, or of a complex molecule, such as 5'-phospho-DNA, through a phosphate group. Target amino acid is usually serine, threonine or tyrosine residues (mostly in eukaryotes), aspartic acid or histidine residues (mostly in prokaryotes).
18 Terima kasihProgram Studi KimiaInstitut Teknologi Bandung2009