活性中心α螺旋連接處突變對兩種倍半萜合酶產(chǎn)物特異性的影響

活性中心α螺旋連接處突變對兩種倍半萜合酶產(chǎn)物特異性的影響摘要：本文通過對α螺旋連接處進行不同的突變，研究了對兩種倍半萜合酶產(chǎn)物特異性的影響。首先，通過構(gòu)建突變搭載載體，轉(zhuǎn)化E.coliBL21（DE3）表達宿主菌，進一步鑒定轉(zhuǎn)化得到的重組表達菌株；其次，采用純化蛋白，進行酶活測定和產(chǎn)物分析，結(jié)果顯示，活性中心α螺旋連接處的突變能夠顯著影響雙重鍵環(huán)的形成和選擇性催化作用；最后，我們對其中一種具有較高活性的突變體進行活性及穩(wěn)定性等方面的比較研究，結(jié)果顯示，活性中心α螺旋連接處的易變性具有較為可控性的特點，可為異構(gòu)化合物液相分離、生物相互作用等相關(guān)領(lǐng)域提供參考。

關(guān)鍵詞：α螺旋連接處；突變；倍半萜合酶；產(chǎn)物特異性；酶活測定；生物相互作用

Abstract:Thispaperstudiestheeffectofmutationsintheα-helicalconnectingregiononthespecificityoftwoterpenesynthases.Firstly,themutationcarrierwasconstructedandtransformedintoE.coliBL21(DE3)expressionhosttofurtheridentifytherecombinantexpressionstrainsobtainedbytransformation.Secondly,purifiedproteinswereusedforenzymeactivitymeasurementandproductanalysis,andtheresultsshowedthatmutationattheα-helicalconnectingregionoftheactivecentercouldsignificantlyaffecttheformationofdoublebondringandselectivecatalyticactivity.Finally,wecomparedtheactivityandstabilityofoneofthemutantswithhigheractivity,andtheresultsshowedthatthevariabilityoftheactivecenteroftheα-helicalconnectionwascontrollable,whichcouldprovidereferenceforliquidphaseseparationofisomericcompounds,biologicalinteractionsandotherrelatedfields.

Keywords:α-helicalconnection;mutation;terpenesynthase;productspecificity;enzymeactivitymeasurement;biologicalinteractioTerpenesynthasesareagroupofenzymesthatareinvolvedinthebiosynthesisofterpenes,adiverseandimportantclassofnaturalproducts.Theseenzymescatalyzetheconversionofsimpleprecursorsintoawiderangeofterpenes,whichareusedinvariousfieldssuchasfragrance,flavor,pharmaceuticals,andbiofuels.

Inourstudy,wefocusedonunderstandingtheroleoftheα-helicalconnectioninthecatalyticactivityandproductspecificityofaterpenesynthase.Throughsite-directedmutagenesis,wecreatedseveralmutantsthattargetedspecificresiduesintheα-helicalconnection,andassessedtheireffectsontheenzyme'scatalyticactivityandproductprofile.

Ourresultsshowedthatmutationsintheα-helicalconnectioncouldsignificantlyaltertheenzyme'scatalyticactivityandproductspecificity.Weidentifiedseveralmutantsthathadhighercatalyticactivityandproduceddifferentproductscomparedtothewild-typeenzyme.Thisdemonstratedthattheα-helicalconnectionplaysanimportantroleindeterminingthecatalyticactivityandproductspecificityofterpenesynthases.

Furthermore,weinvestigatedthestabilityofoneofthemutantsthathadhigheractivity,andfoundthatthevariabilityoftheactivecenteroftheα-helicalconnectionwascontrollable,whichcouldprovidereferenceforliquidphaseseparationofisomericcompounds,biologicalinteractions,andotherrelatedfields.Thissuggeststhattheα-helicalconnectioncouldbeapotentialtargetforenzymeengineeringandoptimizationinthefuture.

Overall,ourstudyprovidesimportantinsightsintothemolecularmechanismsunderlyingthecatalyticactivityandproductspecificityofterpenesynthases,andhighlightsthepotentialoftheα-helicalconnectionasatargetforenzymeoptimizationinvariousapplicationsInadditiontoitspotentialapplicationsinenzymeengineeringandoptimization,thestudyofterpenesynthaseshasbroaderimplicationsfortheunderstandingofnaturalproductbiosynthesisandevolution.Terpenesareoneofthelargestandmoststructurallydiverseclassesofnaturalproducts,withover70,000knowncompoundsproducedbyplants,fungi,bacteria,andanimals.Theyplayimportantrolesinnumerousbiologicalprocesses,includingdefenseagainstherbivoresandpathogens,attractionofpollinatorsandseeddispersers,andcommunicationbetweenorganisms.

Thebiosynthesisofterpenesinvolvestheassemblyofsimpleisoprenoidprecursors,suchasisopentenyldiphosphate(IPP)anddimethylallyldiphosphate(DMAPP),intocomplexstructuresbyterpenesynthases.Theseenzymeshaveevolvedtocatalyzeawiderangeofreactions,includingcyclization,rearrangement,andoxidation,andcanproduceavarietyofproductswithdifferentstereochemistry,regiochemistry,andfunctionalgroups.Thediversityofterpenesynthasesandtheirproductsisthoughttohavearisenthroughgeneduplication,neofunctionalization,subfunctionalization,andcoevolutionwithotherbiosyntheticandregulatoryenzymes.

Thestudyofterpenesynthasescanthusshedlightonthemechanismsandconstraintsofnaturalproductbiosynthesisandevolution,aswellastheecologicalandevolutionaryrolesofnaturalproductsindifferentorganismsandenvironments.Forexample,theidentificationofterpenesynthasesinvolvedintheproductionofpheromones,toxins,andantimicrobialcompoundsininsectsandbacteriacouldleadtothedevelopmentofnewpestcontrolanddrugdiscoverystrategies.Thecharacterizationofterpenesynthasesinmedicinalplantsandfungicouldprovideinsightsintothebiosynthesisandregulationofbioactivecompoundswiththerapeuticpotential.Theengineeringofterpenesynthasesinplantsandmicrobescouldenabletheproductionofnovelandhigh-valueterpenesforindustrial,agricultural,andenvironmentalapplications.

Inconclusion,thestudyofterpenesynthasesisarichandexcitingfieldthatspansvariousdisciplinesandapplications.Bycombiningbiochemical,structural,genetic,andecologicalapproaches,researcherscanuncoverthemolecularmechanismsofterpenebiosynthesis,theevolutionaryhistoryofterpenesynthases,andtheecologicalrolesofterpenesindifferentorganismsandecosystems.Thecontinuedexplorationofterpenesynthasesandtheirproductswillundoubtedlyleadtonewdiscoveries,innovations,andinsightsintheyearstocomeInadditiontotheresearchareasmentionedabove,thestudyofterpenesalsohasnumerouspracticalapplications.Forexample,terpenesarewidelyusedinvariousindustries,suchaspharmaceuticals,flavorandfragrance,andcosmetics.Inthepharmaceuticalindustry,terpenesareusedasstartingmaterialsforthesynthesisofvariousdrugs,suchasartemisinin,whichisusedtotreatmalaria.

Terpenesarealsoanimportantcomponentofessentialoils,whicharewidelyusedinperfumesandaromatherapyproducts.Theuniqueanddiversepropertiesofdifferentterpenesmakethemidealforcreatingspecificscentsandflavors.Forexample,limoneneisacommonterpenefoundinorangeandlemonpeels,whichgivesthemtheircharacteristiccitrusscent.

Furthermore,terpeneshavepotentialapplicationsinagriculture,astheycanactasnaturalpesticidesandrepellents.Forinstance,someterpenesaretoxictoinsectpests,whileotherscanrepelpestsbycreatingastrongodorthatmaskstheplant'sscent.Terpenesmayalsohelpplantsdefendagainstdiseasesandenhancetheirtolerancetoenvironmentalstress.

Overall,thestudyofterpenesisafascinatingandmultidisciplinaryfieldwithnumerousresearchopportunitiesandpracticalapplications.Withadvancesintechnolog