Nitrate Reductase (NADH) EC

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1 Nitrate Reductase (NADH) EC 1.7.1.1
PRESENTASI ENZIMOLOGI KI- 5162 Nitrate Reductase (NADH) EC Oleh: Dita Ayu Pratiwi ( ) Sofi Siti Shofiyah ( )

2 Agenda Presentasi Tata nama enzim Kofaktor Mekanisme katalisis
Manfaat enzim Struktur enzim Tahap isolasi dan pemurnian enzim Aplikasi Industri

3 IUBMB Enzyme Nomenclature EC 1.7.1.1
1 kelompok oxidoreductase, kelompok sub class 7Acting on other nitrogenous compounds as donors Kelompok subsubclass1With NAD or NADP as acceptor Sumber : 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[1]. Sumber: Fischer, et. al The Plant Cell.

6 Cleland Plot NR Sumber: Howard, et. al Journal Biological Chemistry

7 Asimilasi Nitrogen NITRATE 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 NR Sumber: Fischer, et. al The Plant Cell

9 Sisi Aktif Enzim dan Muatan Permukaan NR
Sumber: Fischer, et. al The Plant Cell

10 Arabidopsis thaliana Tanaman berbunga kecil yang hidup di dataran eropa, asia, barat laut afrika Tumbuh di musim semi Mempunyai siklus hidup yang relatif singkat Populer sebagai model organisme untuk memahami biologi molekul banyak tanaman

11 Produksi Rekombinan NR
NR diekspresikan Diekspresikan di methylaptrophic yeast Pickia pastoris strain GS115 dari Arabdopsisi NIA NR cDNA pAtc-4 Pertumbuhan kultur Di kultur 500mL yang mengandung UPD media (1% yeast ekstrak, 2%peptone, dan 2% Glc, a11 w/v) Induksi ekspresi NR Sel 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% metanol Sumber: Wenpei Su, et. al Plan Physiology.

12 Produksi Rekombinan NR (Lanjt)
Kultur sel Ditambahkan methanol 1% (v/v) dan Na-Mo (0,2 mM) setiap 24 jam Setelah 72 jam periode induksi cel dikumpulkan, disentrifuga dan diresuspensi kembali, Diresuspensi Extraction 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 menit Ekstrak kasar Disentrifuga pada g selama 20 menit pada 4oC Diuji aktifitas ekstrak kasar dan dimurnikan Recombinant Arabidopsis NADH:NR Production and Purification NR was expressed in the methylotrophic yeast Pickia pastoris strain GS115 from the Arabidopsis NIA2 NR cDNA pAtc-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-mL cultures containing YPD media (1% yeast extract, 2% peptone, and 2% Glc, a11 w/v) to an A,, of 10. The log-phase cells were washed and then resuspended to an A600 of 5 in 1 L of modified MM media (2.8% [w /VI yeast nitrogen base with ammonium sulfate, 100 mM potassium phosphate, pH 6.0, 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 the culture every 24 h. After a 72-h induction period the cells were collected by centrifugation, washed, and then resuspended in extraction buffer containing 50 mM sodium phosphate, pH 7.3, 1 mM EDTA, and 5% (v/v) glycerol to an A600 of 50 to 100. The cells were broken with a Bead Beater (Biospec Products, Bartlesville, OK) using 0.5-pm glass beads by processing for 5 min using 30-s bursts followed by 1-min cooling periods. The crude extract was then centrifuged at 15,0008 for 20 min at 4°C. NR assays of crude extracts and purified samples were performed as previously described (Campbell and Smarrelli, 1978; Redinbaugh and Campbell, 1985). Sumber: Wenpei Su, et. al Plan Physiology.

13 Permunian NR Sumber: Wenpei Su, et. al. 1997. Plan Physiology.
Ekstrak kasar Pengendapan 30-45% amonium sulfat Pelet dilarutkan padan 50mM Natrium fosfat, ph 7,3, dan 1mM EDTA (bufferA) dialirkan ke Blue-Sepharose Blue Sepharose yang berikatan dengan NR dikumpulkan dengan filtrasi vakum yang dicuci dengan kolom 1,5 cm Kolom kromatografi Dielusi dengan 0,1 mM NADH dalam buffer A One unit of NR activity is defined as 1 pmol of nitrite produced/ min. For the purification of wild-type NR, crude cell extracts were subjected to a 30 to 45% ammonium sulfate precipitation. The resulting pellet was dissolved in 50 mM sodium phosphate, pH 7.3, and 1 mM EDTA (buffer A) and batch-bound to Blue-Sepharose (Campbell and Smarrelli, 1978). The Blue-Sepharose with bound NR was collected by vacuum filtration, thoroughly washed with buffer A, and packed in a 1.5-cm column. The column was eluted with 0.1 mM NADH in buffer A. Fractions containing NR activity were pooled, buffer exchanged, and concentrated with 25 mM Mops (ultrapure grade, Calbiochem), pH 7.0, using a Centriprep 30 concentrator (Amicon, Beverly, MA). Next, the partially purified sample was applied to a 1.5-mL column of 5'-AMP Sepharose (Pharmacia). Sumber: Wenpei Su, et. al Plan Physiology.

14 Pemurnian NR (Lanjt) Sumber: Wenpei Su, et. al. 1997. Plan Physiology.
Fraksi NR Dipekatkan dengan 25mM Mops (ultrapure grade, Calbiochem), pH 7,0 menggunakan Centriprep 30 concetrator Sampe setengah murni Dialirkan ke 1,5 mL kolum 5’-AMP sepharose Kolum 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 7,0 The column was washed with 5 column volumes of 50 mM Mops, pH 7.0, and 1 mM EDTA (buffer B). NR was then eluted with 0.1 mM NADH in buffer B. Fractions containing more than 2 units/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 NR Sampel NR murni Eksperimen 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 Spektrofotometry Konsterasi Subtrat Divariasikan dari 0,5-20 μM untuk NADH dan 2,5-60 μM untuk KNO3 Analisis Kecepatan awal reaksi dianalisis dengan Enzpack software Spektrum dari oksidasi sampel dan NADH(red) ditentukan pada 25oC menggunakan UV/Vis dioda array spectofotometer NR samples purified as described above were used. Kinetic experiments were performed at 25'C in 1 mL of buffer containing 50 mM Mops, pH 7.0, by following the decrease in A,,, using a 1-cm cuvette and a UV/Vis spectrophotometer (model no. 1201, Shimadzu, Kyoto, Japan). The concentration of the substrates varied from 0.5 to 20 p~ for NADH (Sigma) and 2.5 to 60 p~ for potassium nitrate. The results of three initial velocity experiments were analyzed using Enzpack software (Elsevier Biosoft, Cambridge, UK). Spectra of oxidized and NADH-reduced samples were determined at 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 Referensi Wenpei 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 Chemistry Lawrie 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 genes Disulfide 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 kasih Program Studi Kimia Institut Teknologi Bandung 2009