Kvikar hreyfingar og fjölfærni í kuldavirku ensími: tvenndargerð alkalísks fosfatasa - verkefni lokið

Fréttatilkynning verkefnisstjóra

17.1.2019

Almennt séð, er starfsgeta ensíma (sem er einn flokkur próteina) mjög háð markvissum innri hreyfingum í sameindunum, sem aftur er háð ýmsum umhverfisþáttum svo sem hitastigi og sýrustigi. Einnig virðist þetta tiltekna sjávarensím hafa náð að hámarka virkni sína í saltríku umhverfi, líkt og finnst í sjó. Með markvissum skiptum á amínósýrum í próteinum má móta bæði virkni þeirra og stöðugleika, sem aðlagar þau aðstæðum, líkt og gerst hefur í gegnum  þróunarsöguna. Alkalískur fosfatasi er strengur gerður úr 500 samtengdum amínósýrum, sem loða saman og gefa próteininu sitt einstaka hnattlaga svipmót. Prótein eru á sífelldu iði og þessar innri hreyfingar eru m.a. grundvöllur þess hversu ólíkum störfum þau geta gegnt. Þar sem kvikar hreyfingar próteinsameinda eru mjög hraðar, og sameindirnar smásæjar, er erfitt að mæla þær með beinum hætti. Þrátt fyrir það, er þetta eitt af virkustu sviðum próteinrannsókna nú um stundir, og tekist á um ýmsa þætti sem að því lúta. Markmið verkefnisins var að leggja til málanna bæði ný gögn og hugmyndir varðandi samskipti milli undireininga í áðurnefndu ensími með tilraunum og tölvureikningum. Ensímið starfar einungis eðlilega ef tvær eins próteineiningar mynda tvennd, og er því gott líkan fyrir nauðsynleg samskipti og samtal innan próteinklasa, sem eru algengir í frumum. Okkar afbrigði er eitt af kuldavirkustu afbrigðum þessa ensíms sem þekkt eru og bauð það uppá ýmis komar samanburð við minna virk afbrigði úr frá mismun í innri gerð. Loks er ensímið úr fjölskyldu ensíma sem sýnir fjölvirkni, sem þýðir að ensímið getur hvatað önnur hvörf en því er nú ætlað í náttúrunni, en það er líklega bergmál úr árdaga, þegar sértækni ensíma var almennt mun víðari en síðar varð með þróun þeirra. Rannsóknir á slíkum eiginleikum geta aukið skilning á því hvernig breyta má náttúrulegum ensímum í tæki til nota í iðnaði þar sem hvötun nýrra efnahvarfa er þörf.

Í verkefninu var svarað grundvallarspurningum varðandi samspil milli undireininga ensímsins og hvaða efna- og eðlisfræðilegu ferlar liggja að baki aðlögun þess að lágu hitastigi. Niðurstöður renndu stoðum undir þá tilgátu að samskipti undireininga á staðbundnum svæðum ensímsins væru nauðsynlegur grundvöllur fyrir virkni þess með því að miðla kvikum og markvissum hreyfingum innan ensímsins.

English:

The project was focused on a cold-active variant of an enzyme from a marine environment. The enzyme is common in nature and well known in most laboratories (alkaline phosphatase). Mutations in this enzyme cause bone deformations and gut inflammation in humans, whereas in bacteria it takes part in the acquisition of phosphorus ions. Most enzymes function as homodimers or higher order oligomers. The reason for having such large structures is still controversial today and, therefore, a worthwhile endeavor to seek additional experimental facts.

Generally, complex internal motions in proteins/enzymes form an essential part of their amazing functional abilities, and is dependent on environmental factors such as temperature and pH. In our particular case, the enzyme has optimized its functional abilities to high salt conditions, such as those found in the open ocean. Rational replacement of amino acid residues by site-directed mutagenesis can be used the shape the functional and structural properties of enzymes to adapt them to their environments, as has happened through evolutionary history. Alkaline phosphatase is made from 500 amino acid residues linked in a string that adhere to one another and form a certain characteristic spherical conformation. Proteins are dynamic, and these internal motions are a key-element in their abilities to perform all kinds of different tasks. Since protein motions are very rapid and the molecules minute, it has proven impossible to observe these directly. Yet, this field is one of the most active research areas at present, and he role of protein dynamics in enzyme catalysis is also one of the most controversial areas in enzymology today. This project had the main aim of contributing data and ideas to this field with emphasis on the interactions between the subunits in our enzyme using experiments and computer simulations. The enzyme under study is only functional as a dimer of identical subunits, providing a good model to understand better the functional role of protein-protein interactions. Secondly, we were studying one of the most cold-active member of our enzyme family, giving opportunities for comparisons with less efficient enzyme relatives. Lastly, the AP family is a fertile ground for studying the role of catalytic promiscuity, believed to be a remnant of evolution, when enzymes were fewer and had much lesser specificity than today. Studying this is a potential source of knowledge as to how to turn existing enzymes into tools with new functions for practical use in industries.

The project answered some fundamental questions regarding the interactions between subunits in the enzyme and also which chemical and physical processes underlie its adaptation to low temperature. The results supported the hypothesis that local interaction between subunit were a necessary basis for its functions by mediating dynamic and predetermined motions within the enzyme.

As the product of basic research, the results become part of the international scientific community that is working toward a fuller understanding of the functioning of enzymes, nature's very efficient catalysts. Such catalyst will increasingly be used in future industries as knowledge about them increases to tailor them to specific jobs outside what natural organisms have needed. In the first instance, the results will be applied in designing further hypotheses regarding the most basic aspects of catalytic processes in cold-active/oligomeric enzymes and to plan experiments to test those ideas. Enzymes are the main products of the genome that drive biological processes (life) and understanding their properties and interaction with other cell components is a key-element in the development of pharmaceuticals and applications in industries in general. Having the gene sequence is not going to provide such information. The sensitivity of enzymes to all kinds of environmental factors, even simple ones such as temperature, pH and ionic strength (found to be very important in our case), is often overlooked/neglected. Our study has provided a reminder, that in vitro measurements are not necessarily giving the same results as would be observed in the natural environment of a particularly enzyme.

Milestones that were set forth and achieved:

• Better understanding of how to design stronger or looser intersubunit interactions into VAP (and thus other similar protein-protein complexes).
• Answer the question of how does the oligomeric state affect reaction kinetics.
• Better understand the role of divalent cations in maintaining the dimeric state and driving catalysis.
• Make new variants of AP better suited for practical use. The design criteria: Increased thermostability and tighter metal binding without impairment of activity.
• Basic new understanding of principle of cold-adaptation in enzymes with potential industrial benefits.
• New X-ray structures of VAP variants.
• One Ph.D. degree thesis. One to two M.Sc. theses. Five B.Sc. practical projects. All in the public domain with open access.
• Young investigators given the opportunity to train for future work in academia, in the public sector, or private industries and services.
• New computer algorithms created for general use.
• Papers in widely read refereed journals detailing extensive characterization of VAP (3-4).

Heiti verkefnis: Kvikar hreyfingar og fjölfærni í kuldavirku ensími: tvenndargerð alkalísks fosfatasa / Dimer dynamics and promiscuity in a cold-active enzyme: The case of alkaline phosphatase
Verkefnisstjóri: Bjarni Ásgeirsson, Raunvísindastofnun
Tegund styrks: Verkefnisstyrkur
Styrktímabil: 2014-2016
Fjárhæð styrks: 28,502 millj. kr. alls
Tilvísunarnúmer Rannís:  141619