Difference between revisions of "Part:BBa K1998013"

Latest revision as of 02:37, 21 October 2016

MgPPIX to Chlorophyll a plasmid

Sequence and Features

Overview

This composite part is one of two plasmids assembled for the chlorophyll biosynthesis pathway. It is composed of yCF54, chlM, pOR, dVR1, chlP and chlG. These genes code for the proteins Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase (yCF54), Magnesium-protoporphyrin O-methyltransferase (chlM), Light-dependent protochlorophyllide reductase (pOR), 3,8-divinyl Geranylgeranyl reductase (chlP), protochlorophyllide a 8-vinyl reductase (dVR1) and Chlorophyll synthetase (chlG).
These genes form the back end of the chlorophyll biosynthesis pathway and are responsible for the conversion of divinyl protochlorophyllide (the precursor of chlorophyll a after all enzymes react with protoporphyrin IX from the genes of Mg-chelatase plasmid [BBa_K1998000]) to chlorophyll a.

Biology & Literature

The first gene, yCF54, works with the cTH1 gene and Plastocyanin within the oxidative cyclase pathway. It's interaction with other genes results in the catalysis of the biosynthesis pathway from Mg-protoporphyrin IX to Protochlorophyllide [1]. Deletion studies have detected the importance of yCF54 in maintaining levels of Mg-protoporphyrin IX methyl ester indicating that the yCF54 gene is critical to both the assembly and function of the cyclase complex [1].

The second gene in this part is chlM. chlM encodes for a magnesium protoporhyrin IX methytransferase. chlM's role in the pathway is to methylate Mg-protoporphyrin IX which then aids the catalysis of Mg-cheltase [2]. This then facilitates the formation of the final protochlorophyllide. Involved in the second step of the pathway, the chlM gene is involved in the transfer of a methyl group onto one of the rings of magnesium protoporphyrin which forms the magnesium prootoporhyrin IX monomethylester [3, 4].

The pOR gene is a light dependent protochlorophyllidereductase. It's role is to convert protochlorophyllide to chlorophyllide using NADPH and light as the reductant. Formation of the POR protein in plants is important as without it the chloroplasts do not function [5, 6]. The protochlorophyllide reduces to chlorophyllide by being excited through light functioning as a catalysis. The excitation of the pOR molecule and therefore the transformation of it to chlorophyllide cause the conversion of the membranes from an inactive state into active chloroplast [7]. Through this transformation into active chloroplasts, energy is produced and now able to be used by the plant.

dVR1 encodes for an enzyme, 3,8-divinyl protochlorophyllide 8-vinyl reductase, which reduces divinly protochlorophyllide a to a monovinyl protochlorophyllide a [8]. By reducing the protochlorophyllide, pOR can convert it to chlorophyllide.

The chlP gene encodes for an enzyme, geranylgeranyl reductase. This enzyme adds the geranylgeranyl pyrophosphate chain to the chlorophyllide molecule. Through hydrogenation of geranylgeranyl diphosphate (GGPP), it reduces the GGPP's double bonds [9]. This allows the synthesis of chlorophylls another components of the pathway.

The final gene in this part is chlG which encodes the enzyme chlorophyll synthetase. This enzyme catalyses the esterification of chlorophyllide with GGPP, allowing for the production of chlorophyll.

Part Verification

Fig 1. Gel electrophoresis (1% agarose) provides evidence of successful assembly of Mg-PPIX to Chlorophyll a plasmid (6.3 kbp) (Lane 5). Plasmid was assembled via 3A assembly and double digested to reveal the biobrick backbone (2000 bp) and the correct insert size.

Protein information

YCF54
Mass: 17.08kDa
Sequence: MAPAAASADKATAAEYYALVCNAEWFFMDPQNESVAEQLREKVRFFKEQNKERDFFIVPNPKWLDAKFPEQAKQVKRPCVALVSTDKMWITFMKLRLDRVLKIDLKSM PASEVLAAGEALPDFKPDGKWTAPYARYTPGWWNVFLPNH

ChlM
Mass: 30.45kDa Sequence: MASEIAQTADVGSLTFAVGGVGAVVGLGALLVATDHQKRRSEQMKSFDGDEKEAVKDYFNTAGFERWRKIYGETDEVNKVQLDIRTGHAQTVDKVLRWVDEEGSVQGIT VADCGCGTGSLAIQLALRGAAVSASDISAAMASEAEQRYQQAVAAGQGKAPKVAPKFEALDLESVKGKYDTVTCLDVMIHYPQDKVDAMITHLAGLSDRRLIISFAPKTL SYSILKRIGELFPGPSKATRAYLHREEDVEAALKRAGFKVTKREMTATSFYFSRLLEAIRE

POR
Mass: 41.87kDa
Sequence:
MVVCAATATAPSPSLADKFKPNAIARVPATQQKQTAIITGASSGLGLNAAKALAATGEWHVVMACRDFLKAEQAAKKVGMPAGSYSILHLDLSSLESVRQFVQNFKASGR RLDALVCNAAVYLPTAKEPRFTADGFELSVGTNHLGHFLLTNLLLDDLKNAPNKQPRCIIVGSITGNTNTLAGNVPPKANLGDLSGLAAGVPAANPMMDGQEFNGAKAYK DSKVACMMTV RQMHQRFHDATGITFASLYPGCIAETGLFREHVPLFKTLFPPFQKYITKGYVSEEEAGRRLAAVISDPKLNKSGAYWSWSSTTGSFDNK

DVR1
Mass: 37kDa
Sequence:
MAMAASRQAVRVAAAVDADYRKREPKDVRVLVVGPTGYIGKFVVKELVSRGYNVVAFARENAGIKGKMGREDIVKEFHGAEVRFGSVLDPASLRDVAFKDPVDVVVSCLA SRTGGKKDSWLIDYTATKNSLDVARASGAKHFVLLSAICVQKPLLEFQKAKLQFESDLQAAGDITYSIVRPTAFFKSIAGQIDIVKKGNPYVMFGDGNLAACKPISEADLASF IADCVTEQNKVNKVLPIGGPSKAFTAKQQADLLFNITGLPPKYFPVPVALMDGMIGLFDSLAKLFPQLEDSAEFARIGKYYATESMLVYDEARGVYRKTKRLVTARTRWKTS SLVQ

ChlP
Mass: 47kDa
Sequence:
MVIGGGPSGACAAETLAKGGVETFLLERKLDNCKPCGGAIPLCMVEEFDLPMEIIDRRVTKMKMISPSNREVDVGKTLSETEWIGMCRREVFDDYLRNRAQKLGANIVNGL FMRSEQQSAEGPFTIHYNSYEDGSKMGKPATLEVDMIIGADGANSRIAKEIDAGEYDYAIAFQERIRIPDDKMKYYENLAEMYVGDDVSPDFYGWVFPKYDHVAVGTGTVVN KTAIKQYQQATRDRSKVKTEGGKIIRVEAHPIPEHPRPRRCKGRVALVGDAAGYVTKCSGEGIYFAAKSGRMAAEAIVEGSANGTKMCGEDAIRVYLDKWDRKYWTTYKVLD ILQKVFYRSNPAREAFVELCEDSYVQKMTFDSYLYKTVVPGNPLDDVKLLVRTVSSILRSNALRSVNSKSVNVSFGSKANEERVM AA

ChlG
Mass: 36.84kDa
Sequence:
MNQQATEEKSDTNSAARQMLGMKGAALETDIWKIRVQLTKPVTWIPLIWGVACGAAASGHYQWNNPTQIAQLLTCMMMSGPFLTGYTQTINDWYDREIDAINEPYRPIPS GRISERDVIVQIWVLLLGGIGLAYTLDQWAGHTTPVMLQLTIFGSFISYIYSAPPLKLKQSGWAGNYALGSSYIALPWWAGQALFGTLTLDVMALTIAYSLAGLGIAIVNDFKSI EGDRQ MGLQSLPVAFGVDTAKWICVSTIDVTQLGVAAYLAWGLHEELYGAVLLALILPQIYFQYKYFLPDPIANDVKYQASAQPFLVFGLLTAGLACGHHVNAVAA AASAAGAL

References

[1] Hollingshead S, Kopečná J, Jackson PJ, Canniffe DP, Davison PA, Dickman MJ, Sobotka R, Hunter CN. Conserved chloroplast open-reading frame ycf54 is required for activity of the magnesium protoporphyrin monomethylester oxidative cyclase in Synechocystis PCC 6803. Journal of Biological Chemistry. 2012 Aug 10;287(33):27823-33.

[2] Alawady A, Reski R, Yaronskaya E, Grimm B. Cloning and expression of the tobacco CHLM sequence encoding Mg protoporphyrin IX methyltransferase and its interaction with Mg chelatase. Plant molecular biology. 2005 Mar 1;57(5):679-91.

[3] Shepherd M, McLean S, Hunter CN. Kinetic basis for linking the first two enzymes of chlorophyll biosynthesis. FEBS Journal. 2005 Sep 1;272(17):4532-9.

[4] Meinecke L, Alawady A, Schroda M, Willows R, Kobayashi MC, Niyogi KK, Grimm B, Beck CF. Chlorophyll-deficient mutants of Chlamydomonas reinhardtii that accumulate magnesium protoporphyrin IX. Plant molecular biology. 2010 Apr 1;72(6):643-58.

[5] Fujita Y. Protochlorophyllide reduction: a key step in the greening of plants. Plant and cell physiology. 1996 Jun 1;37(4):411-21.

[6] Darrah PM, Kay SA, Teakle GR, Griffiths WT. Cloning and sequencing of protochlorophyllide reductase. Biochemical Journal. 1990 Feb 1;265(3):789-98.

[7] Griffiths WT. Characterization of the terminal stages of chlorophyll (ide) synthesis in etioplast membrane preparations. Biochemical Journal. 1975 Dec 1;152(3):623-55.

[8] Davies K, editor. Annual plant reviews, plant pigments and their manipulation. John Wiley & Sons; 2009 Feb 12.

[9] Shpilyov AV, Zinchenko VV, Shestakov SV, Grimm B, Lokstein H. Inactivation of the geranylgeranyl reductase (ChlP) gene in the cyanobacterium Synechocystis sp. PCC 6803. Biochimica et Biophysica Acta (BBA)-Bioenergetics. 2005 Feb 17;1706(3):195-203.

[10] Tamiaki H, Shibata R, Mizoguchi T. The 17‐Propionate Function of (Bacterio) chlorophylls: Biological Implication of Their Long Esterifying Chains in Photosynthetic Systems. Photochemistry and photobiology. 2007 Jan 1;83(1):152-62.