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[Music]

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okay so pictured here is a cartoon of

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the modern nitrogen cycle so nitrogen is

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an essential element for life we need it

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for DNA we need it for proteins but even

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though our atmosphere is almost entirely

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composed of nitrogen most organisms

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can't use it in this form so nitrogen

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needs to be reduced or fixed and this

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process is carried out by a small

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selection of microbes that basically

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provide all of the fixed nitrogen nearly

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all of the naturally fixed nitrogen to

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the biosphere so biological nitrogen

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fixation is also an exceedingly ancient

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metabolic process and it originated with

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the evolution of the nitrogenase enzyme

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so this is the only enzyme that's

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capable of fixing nitrogen and it

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evolved somewhere between two and three

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or more billion years ago and so really

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it's the activity of this nitrogen

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fixing enzyme nitrogenase that has

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helped to sustain the biosphere over

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potentially nearly half or also perhaps

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the majority of Earth's history

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alright so pictured here on the left is

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the nitrogenous protein complex

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nitrogenous is also interesting because

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it's a metallo enzyme and that means

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that it incorporates metal clusters in

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order to catalyze the reduction of

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nitrogen I'm so pictured here this is

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the cofactor in this little insert image

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and this particular cofactor is called

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the iron molybdenum cofactor and it's

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called that because it incorporates one

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unique atom of molybdenum that's the the

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kind of teal colored circle but there

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are other forms of nitrogenous and so

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the molybdenum four is by far the most

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common it's also the most efficient but

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there are also two other rarer forms so

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they instead incorporate an atom of

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vanadium or an additional atom of iron

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in place of molybdenum and so

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phylogenetic analyses have established

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that each of these three nitrogenous

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forms are genetically distinct but they

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are evolutionarily related and so then

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that presents the question well what was

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the metal usage of ancestral

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nitrogenases and so the reason why this

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question is particularly interesting

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from an astrobiological perspective is

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it kind of requires that we probe this

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potential ancient and interaction

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between

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marine geochemistry through time as well

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as the evolution of nitrogen fixation

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and other metabolic processes so these

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three metals that are incorporated in

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the nitrogenous cofactor are themselves

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redox sensitive and so their abundances

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in in the Earth's oceans through time

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have changed largely as a function of

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Earth's surface oxygenation so for

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example molybdenum is one example its

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abundance in the Earth's oceans has was

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probably not very high until after the

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great oxidation event and so this

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observation then led to the hypothesis

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that perhaps ancestral forms of

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nitrogenase that originated prior to the

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great oxidation event maybe these were

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in fact molybdenum independent because

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there wasn't enough molybdenum to go

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around basically still more recent

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phylogenetic analyses have instead

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suggested that the molybdenum form

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preceded the evolution of the vanadium

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and iron forms and maybe this didn't

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happen until after the great oxidation

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event or somewhere close to it so we

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wanted to further explore this history

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of nitrogenous metal usage and we wanted

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to specifically be able to infer the

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metal dependence of reconstructed

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ancestral nitrogenous enzymes and so we

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use a method called ancestral sequence

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reconstructions so basically this

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entails collecting a modern data set of

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in this case nitrogenase protein

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sequences using that information to

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build a phylogenetic tree and then using

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statistical methods to then infer an

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ancestral sequence that corresponds to

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some ancestral node of interest and then

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with that sequence you can analyze it

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for interesting features and you can

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also use it to model ancestral protein

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structures and so what we wanted to do

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is we wanted to use this method to

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specifically analyze the active site

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sequence and structural features that

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had previously been used to classify the

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metal dependence of modern nitrogenases

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and instead apply it to our inferred

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ancestors so pictured here on the left

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this is our nitrogenase phylogenetic

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tree so this was built from a

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concatenated data set of molybdenum

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vanadium and the iron nitrogenous

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protein sequences oops so most of the

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the tree is made up of molybdenum

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dependent forms I mentioned this these

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are the most common but up here on the

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top this lineage this is where the iron

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and vanadium nitrogenous forms cluster

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um and what's also interesting is they

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seem to branch after the divergence of

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this purple group here at M BMC so this

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is another smaller group of molybdenum

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dependent nitrogenases and so this

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topology seems to be consistent with the

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idea that iron and vanadium forms

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evolved after the evolution of

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molybdenum usage but at the base of this

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lineage or to other claves so there's

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one in yellow one in green these are

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quote-unquote uncharacterized

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nitrogenases and so they're called this

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because we don't have really any

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experimental evidence or any any

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assessment of what metals that these

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nitrogenases incorporate and so they're

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a little bit of a mystery and so along

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this phylogeny

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transect we might expect some kind of

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historical change in metal usage and so

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it's here where we specifically

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reconstructed and analyzed ancestral

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nitrogenase sequences in order to

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basically track this history of metal

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usage ok so the first thing we wanted to

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look at was the active site sequence

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composition and see if we could find

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anything that appeared to correlate with

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metal binding so on the bottom these are

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pairwise loops these are pairwise

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comparisons of extant nitrogenous forms

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against each other it's a heat map so

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the lighter colors indicate that the

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active site sequences lower identity the

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darker colors indicate higher sequence

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identity and so you can see that these

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comparisons do a decent job at

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differentiating the molybdenum forms

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from the vanadium or iron forms and so

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we then bring that up to the top portion

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of this plot where we're comparing the

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ancestors that's a through e with the

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modern forms and we find that most of

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the ancestors B through e seem to look a

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lot more like the molybdenum forms than

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the iron and vanadium forms perhaps with

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the exception of ancestor a at the top

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which happens to be the direct ancestor

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of the iron vanadium forms and so it's

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resemblance to those modern nitrogenase

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is quite reasonable

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all right so we also use these sequences

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to generate models of these ancestor

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ancestral proteins so here they are

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aligned and superimposed on one another

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but we were specifically interested in

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calculating the volume of the active

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site pocket and the reason we were

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interested so that's what's pictured in

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pink right there the reason we were

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interested again was that this parameter

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was previously used to classify metal

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dependence of modern nitrogenases again

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we wanted to apply this to the ancestors

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okay so in this rather complicated plot

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these are pocket volumes both for

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ancestral nitrogenases and for extant

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nitrogenases and i just want to draw

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your attention to the right side for the

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the modern ones and what was kind of

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surprising was that we see that the

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vanadium nitrogenases as well as one

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major group of the molybdenum dependent

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ones seem to occupy a very similar

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volume range in fact they're so similar

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and nearly indistinguishable that

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they're actually more similar to each

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other than are the two different groups

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of molybdenum nitrogenases also

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problematically as it seems like a lot

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of our ancestors also occupy the same

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range and so kind of in contrast to a

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previous study we don't seem to see any

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way to use this information to infer

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metal usage particularly with regard to

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our ancestors alright so the last thing

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we did was we were interested in the

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distribution of proteins that today are

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involved with the synthesis of the

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molybdenum cofactor in nitrogenase and

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so these two proteins to two of the

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various proteins that are involved in

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this pathway NIF II and if n so we

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mapped their distribution on taxa that

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are represented represented in our

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phylogeny and what we found is that

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these two kind of strange clays the

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yellow and green on characterized

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nitrogenases they seem to have either a

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non-existent or incomplete set of NIF E

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and if N and so by that information you

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might infer that they are unable to use

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molybdenum perhaps parsimoniously we

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might also infer that the oldest

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ancestors may similarly not have nafion

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if n and so may

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maybe also molybdenum in the pendant

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okay so as a quick recap it seems like

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our oldest ancestor active site sequence

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compositions resemble the molybdenum

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ones more than the vanadium and iron

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ones but we have this seemingly

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contradictory observation that the basal

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and characterize nitrogenases lack the

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associated proteins for molybdenum

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dependence so let's you solve this

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discrepancy is maybe two main options

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that we can go with perhaps these oldest

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ancestors did in fact use molybdenum and

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these uncharacterized clades may have

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secondarily lost these cofactor proteins

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or maybe they use a different pathway

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for molybdenum usage that we currently

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don't know anything about the second

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option is that perhaps the oldest

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ancestors did not use molybdenum because

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they were not able to synthesize the

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molybdenum cofactor they didn't have

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enough a they didn't have enough n and

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the sequence resemblances that we

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observe may not be entirely definitive

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and that molybdenum usage really did

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evolve with Nephi and if n once

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molybdenum became available and not

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until after the divergence of these

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uncharacterized clades so for a few

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reasons we prefer the second scenario of

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the first thing is that we actually do

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have isotopic signatures of nitrogenous

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activity in the geologic record so these

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were recently reported and they date

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prior to our estimates for marine

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molybdenum availability and they're also

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dated older than the molecular molecular

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clock ages and if EMF in a second it's

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been previously proposed the

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nitrogenases prior to the evolution of

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Na Fianna fen may still have been

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functional so able to fix nitrogen and

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this model kind of still leaves some

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room that could be possible that early

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molybdenum usage may have been may have

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been doable by some different currently

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unknown pathway so we can illustrate

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this model on our phylogeny

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so first potentially we started with a

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nitrogenous that had no associated NIF

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Ian offend proteins therefore was not

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molybdenum dependent at least not in the

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typical way and that this ancestral

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state is still exhibited by

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uncharacterized clades that still exists

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in the

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hello then we have this stepwise

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addition of these co factor proteins

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perhaps starting with NIF e and this is

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still reflected by the uncharacterized

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clades in green and it wasn't until we

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have both Nephi and NIF and that today

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our harbored by the molybdenum dependent

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forms in purple and also in blue that we

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had actual true molybdenum usage and

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then finally it wasn't after until after

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the evolution of molybdenum usage that

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we get the diversification of the iron

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and in vanadium forms alright so in

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conclusion it seems like our ancestors

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at least just looking at the sequences

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seem to resemble molybdenum nitrogenases

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but our phylogenetic analyses suggest

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that canonic canonical of molybdenum

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usage evolved with a cofactor synthesis

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mechanism Nephi and a fen and probably

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not until after the divergence of these

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uncharacterized clades and finally I

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just want to highlight that even though

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the work I'm presented here today is

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entirely computational this method of

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ancestral sequence reconstruction also

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permits us to synthesize their encoding

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genes and quote-unquote resurrect these

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enzymes in the laboratory and so we can

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continue to experimentally test these

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hypotheses so please stay tuned for

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future work ok so to just and here I'd

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like to acknowledge our research group

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at the University of Arizona

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particularly my PI Batool Kjar please

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visit our website if you're interested

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in lots of other fun projects we're

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working on and I'd like to acknowledge

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our funding sources in particular the

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NASA Astrobiology postdoc program so

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thank you very much

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[Music]

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we have time for a couple of questions

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yeah a gene families are sometimes put

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it just super gene families if you look

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deeper and you've lower your homology

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threshold and I'm wondering have you

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looked at a possibly related gene family

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to the nitrogenases that you looked at

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and if so that would give you an

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out-group for your tree yes so so our

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tree is rooted with an out-group in this

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case we used proteins that are they're

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called light independent proto

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chlorophyll I'd reductases they're

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involved with the synthesis of bacteria

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chlorophyll basically and so they do

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share distant homology there's also

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other enzymes that seem to be fairly

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related those are called nitrogenous

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like enzymes and as of yet I don't think

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we know exactly what they do but they

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seem to be in methanogens do they have

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any metal cofactor

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yes they do have metal cofactors they do

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not look very much like the nitrogenous

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cofactor I can't describe it to you off

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the top of my head but yes they are

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metallo enzymes yeah is what another

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possibility be that the ancestral

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nitrogenase sort of could use multiple

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cofactors multiple enzymes and that

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instead in that what you're seeing is

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sort of specializing and canalization to

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being assembled in particular ways and

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maybe getting better as a result yeah so

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we have thought about this kind of

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interesting idea of like an ancestral

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generalist enzyme and it's kind of cool

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to think about it's it's a little bit

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difficult to reconcile with with the

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modern processes of building that

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cofactor which you know rely on a kind

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of very hierarchical structure of

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associated proteins that do the job

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basically and so one potential option is

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that there was only a small subset of

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those proteins that directed perhaps

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just the core of the cofactor deficient

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of molybdenum maybe deficient of

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vanadium but it's I'm not sure of any

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way that you could sort of get other

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metals transiently associating

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the cofactor but maybe by looking at

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these other uncharacterized nitrogenases

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we can sort of expand our idea of the

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possibilities of how this nitrogenase

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could have functioned all right all

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right if there are no other questions

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let's thank again our speakers