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Dark ‘Mirror Organisms’ and Chiral Reciprocity

April 19, 2011

In the previous post, I discussed the notion of the possible existence of a ‘shadow biosphere’ on this planet, inhabited by organisms radically different to the whole lineage of organisms familiar to science. A whole category of ‘shadow life’ (previously referred to as Life-2.0) lies in the potential for ‘inverse chirality’ in living organisms. This theme will be expanded in the present post.

Living Stereochemistry

It is often the case that alternative combinations of an identical set of atoms can form chemical bonds in distinct ways, resulting in chemical isomers. If two compounds are isomeric to each other purely due to the spatial arrangement of the atoms (rather than differing types of chemical bonds), we refer to such a pair as stereoisomers. (Broadly speaking, stereochemistry itself is the general study of the three-dimensional structures of molecules, as indicated by its Greek route stereos, or solid.)

For a simple carbon compound of the formula CX4 (where X = any chemical group bonded to the Carbon atom), the X groups structurally define the corners of a tetrahedron. And if each ‘X’ is a different group, two interesting stereoisomers are possible, where the alternatives are mirror images of each other. As shown in the above ‘mirror’ diagram (with a central carbon atom bonded with groups α-δ), such compounds are not superimposable on each other, and therefore are not physically identical, despite their great chemical similarity. Molecules of this type (D– and L– in the above diagram) are also referred to as chiral.  In this representation, the D– and L– designations are arbitrary, but the same labels are used to refer to the known absolute stereochemical configurations of specific molecules.

Chirality is an inescapable and profoundly important aspect of carbon-based life. Of the important classes of chiral compounds in living biosystems, typically one finds L-amino acids (the building blocks of proteins) and D-sugars, although there are certainly exceptions (as noted below). With respect to the latter sugars, D-ribose is a constituent of the backbone of all nucleic acids, and D-glucose is a fundamental energy source. The specific stereochemistry of biomolecules is critical for correct biochemical interactions. For example, proteins binding to D-sugars with great specificity will fail to recognize their L-isomers, owing to the distinct difference in their stereochemical shapes.

You might well ask, as many others have done, why biology should be arranged in just this manner? Why not D-amino acids and L-sugars – or some kind of mixture? (Proteins composed of some kind of amalgam of both L-and D-amino acids). Suffice it say that although the answer remains elusive, there has been no shortage of conjecture and hypotheses. But there have also been some tantalizing clues from natural sources. For example, amino acids have been detected in certain carbonaceous meteors, but with an unaccounted excess of L– amino acid stereoisomers. For carbon compounds formed through natural processes, a balanced composition of such stereoisomers (a ‘racemic’ mixture) would be expected, so findings of pronounced imbalances in favor of L– forms are especially interesting. Such data give hints that the existing chiral composition of life on this planet is not a ‘frozen accident’ (another popular view) but was at least tipped towards the existing chiral patterns by physical factors whose nature remains to be properly defined. But even if the chirality of Life-1.0 (our familiar life) was the most probable outcome, the possibility of alternative biological chiralities still remains….

Inverse Biology

Following the above very brief elementary background, we can return to the relevance of chirality to the theme of biological ‘dark matter’ (or ‘Life-2.0’). The basic premise is quite simple: what if an alternative biology arose during the earliest stages of molecular evolution, with a distinctly altered pattern of molecular chirality relative to that found in familiar life? And what if at least a vestige of this version of ‘Life-2.0’ persisted until modern times? A number of possible scenarios with alternative bio-chiralities could be envisaged, but a simple version is ‘inverse biology’, where D-amino acids and L-sugars are the norm.

The general idea of ‘inverse-life’ itself has quite a respectable vintage, and has been an oft-used theme in science fiction. A plot can be summarized along the following lines: The chief protagonist encounters some kind of weird physical process whereby the normal molecular chirality of his body is ‘flipped’ or ‘inverted’ (Almost always it is a ‘he’ in such stories, which tend to date from times with little gender-sensitivity).  All is still fine at first, but soon the victim of this ‘inversion’ encounters a sinister problem: despite eating copious amounts of normal foods, he is starving. His new-formed biosystem cannot use foodstuffs of normal chirality, and only items with chiralities that match his own can be used as energy and building materials for his body. So our hero must either find a way to invert his inversion (so to speak), or rustle up an adequate supply of D-amino acids and L-sugars……

Since this sci-fi inversion event involves the creation of a biosystem with molecules with mirror-image stereochemistries, any Life-2.0 version with such features might be termed ‘mirror biology’ with respect to regular Life-1.0. Following the coining of the term ‘spiegelmers’ (of which more below ), we might continue to use the spiegel– root to refer to any such a hypothetical ‘inverted’ organism as a form of ‘spiegel-leben’ (German for mirrorlife), as depicted for the above mirror-feline. One might then collectively liken such postulated spiegel-leben to metaphorical forms of biological anti-matter. Unlike physical anti-matter, though, obviously there is no prospect of these life-types annihilating each other upon physical contact. But could the opposite occur – could they co-exist in close proximity without influencing each other?

And this is the central issue which takes us beyond mere science-fiction – or at least into the realm a testable hypothesis. Unlike the sci-fi scenarios, the notion that a ‘shadow’ biological world with inverted chirality to the norm might exist all around us does not seem to have been seriously entertained until quite recently. The physicist Paul Davies (noted in previous ‘dark matter’ posts  in this general context) has taken this idea with sufficient seriousness to encourage biologists to collaborate in a project testing for the presence of such ‘weird’ organisms in various environmental samples. To do this, one simply proposes that inverse Life-2.0 would require comparable basic building blocks as for regular Life-1.0, but with switched chirality. So suitable media are formulated and sterilized, and then carefully inoculated with the desired samples. It would be expected, of course, that familiar life could not grow, since the proper nutrient materials are not present in the correct stereochemical form. But if the putative inverse-organisms are indeed present, they might well able to use what has been provided and propagate themselves. If significant, such growth would soon become apparent to even the unaided eye by the appearance of turbidity in the otherwise clear defined medium. It’s important to note, though, that failure to grow anything under such circumstance tells you nothing. Even if the potential was there, growth might fail for many reasons. In the previous post it was noted that only a small portion of conventional soil (and other environmental) bacteria can be induced to grow under laboratory conditions, so failure with ‘aliens’ with seem even more likely. (Many Life-1.0 organisms require special nutrients in addition to basic chemical precursors of biomolecules, so there is no reason to suppose that Life-2.0 should be different). But just as familiar life is incredibly diverse, so too could be its hypothetical chirally inverse counterpart. And it would only take one species of inverse-bacteria to show positive growth to create a scientific sensation.

So tests along these lines were set up….and growth was obtained! ….But not weird life.  The bacteria growing under such conditions were using chirally inverse carbon sources, but there was nothing inherently unusual about their general molecular make-up. So what molecular trick allowed them to propagate themselves? These organisms possessed enzymes called racemases, which enable the conversion of one amino acid stereoisomer into its chirally opposite form (or likewise with sugars). Equipped with these, a soup of simple organic compounds with the ‘wrong’ chirality still yields a handy source of nutrients, which such versatile bacteria can use for all their carbon-based needs.

This is an important observation, since it can be used as a springboard to consider how conventional Life-1.0 and inverse Life-2.0 might interact with each other. Recalling the previous post’s discussion of orthogonality, it bears heavily on the question as to whether chirally inverted life-types could remain orthogonal to each other. But first, a couple of background areas should be briefly considered….

D-Amino Acids in the L-Amino Acid World

With the benefit of hindsight, the growth of certain ‘regular’ bacteria in media containing compounds with inverse chirality is predictable. It has long been known that D-amino acids are widely encountered in the prokaryotic world, with an important example being the use of D-alanine as a component of peptidoglycan, the structural material for the cell walls of most bacterial species. And in order to make the necessary D-alanine (and various other D-amino acids), specific racemase enzymes are required. Of course, specialized bacterial uses of various D-amino acids should not detract from the fact that, as with all Life-1.0 on this planet, proteins synthesized by the standard ribosomal machinery are composed exclusively of L-amino acids.

But applications for D-amino acids by Life-1.0 organisms certainly do not stop there! Despite the initial belief that bacteria had a monopoly in this area, D-amino acids have now been found widely distributed among the kingdoms of life. Notably, D-serine is found in significant amounts in mammalian neural tissue, where it has an important physiological role in neurotransmission. (The aberrant production or turnover of D-serine has been implicated in a number of human neurological disease states). The required reserves of D-serine are produced from L-amino through the agency of serine racemase enzymes.

But interesting as this may be, what relevance do such phenomena have towards hypothetical inverse Life-2.0? One more piece of background information is now required…..

 Chiral Reciprocity

In thinking about the stereochemical changes which can be wrought by enzymes, we are of course normally referring to catalytic proteins composed of specific sequences of L-amino acids. Versatile as they are, a particular enzyme itself made up of L-amino acids can thus recognize and bind a target single L-amino acid (its substrate) and convert to into the corresponding D-amino acid.

But what would happen if you made another enzyme, with exactly the same sequence-string of amino acids, except that they were all with opposite chirality? (the D-configuration). An interesting thing, as it turns out. This D-enzyme would be predicted to bind the D-amino acid counterpart to the substrate for the normal enzyme, and then convert it into the corresponding L-amino acid. This kind of ‘swap-over’ effect is known as chiral reciprocity, as depicted in the figure below.

In the situation portrayed by this figure, the initial normal L-enzyme is acting on a short peptide string of D-amino acids to produce a specific modification upon it. The ‘mirror image’ D-enzyme then performs a chirally reciprocal operation by acting on a corresponding L-amino acid peptide, to produce the equivalent modification.

This kind of effect has been shown to work with real enzymes, by the use of whole protein syntheses with D-amino acids. The chiral reciprocity principle has also been used for an interesting application of functional nucleic acids. Specific single-stranded RNA can be selected to bind chiral targets such as amino acids or peptides. Although potentially very useful for drug purposes, this activity is greatly limited by the sensitivity of RNA to rapid enzymatic degradation in vivo. But artificially altered RNA molecules whose ribose sugar moieties are all L-configuration instead of the natural D-form fail to be recognized by such enzymes, and are accordingly very resistant to break-down. Unfortunately, it is hard to predict from first principles the exact sequence of an RNA molecule with desired binding properties. RNAs binding specific ligands can be functionally selected from large libraries of variants – but this cannot be done directly with the unnatural L-forms.

So, a nice trick is to first select a normal D-form RNA (an aptamer) which binds a target molecule with the opposite chirality to what is finally desired (for example, against a D-amino acid or peptide). Then, having determined the sequence of this selected functional RNA, its chirally opposite L– form is synthesized. And by the principal of chiral reciprocity, this novel synthetic L-form RNA should bind the same target molecule as before, but now with inverted target chirality (thus, it will bind a normal L-amino acid if the initial binding target for selection was the corresponding D-amino acid). Such mirror-binders have been referred to as spiegelmers, hence rationalizing the coining of ‘spiegel-leben’ as above.

So, now to put some of these strands together…

Orthogonality and Chiral Reciprocity

Enzymatic reactions are in principle reversible. In the above figure (using the example of serine racemases) arrows are shown going in either direction to indicate the left-to-right (‘forward’) or right-to-left (‘reverse’) reactions. But reversibility does not necessarily equate with forward and reverse equivalence in terms of enzyme kinetics (the rate of reactions and the affinity of enzymes for substrates). In these examples, the rightward arrows are dark to indicate that the forward reaction is favored under physiological circumstances.

Now if we look at the first of these three serine racemases, we see that the production of D-serine from L-serine is the favored forward reaction, in common with the known mammalian enzyme. But it is likely that a little tinkering with racemase-1 could alter its kinetic parameters such that the reverse reaction is favored, as with racemase-2 of the above diagram. (Racemase-2 would have a higher affinity for L-serine as a substrate than D-serine, the opposite of racemase-1).

Then let’s invoke chiral reciprocity once more. From the protein sequence of racemase-2 we could derive a corresponding ‘mirror’ racemase-3, all in the D-amino acid configuration. We would then expect racemase-3 to recognize L-serine, with favored production of D-serine.

And the central issue of interest: If inverse ‘shadow’ Life-2.0 organisms possessed racemase-3, they could exploit amino acids (and proteins) derived from the conventional Life-1.0 world. Of course, a Life-2.0 serine racemase might have a quite distinct sequence from our racemase-3, but that is not the essential issue. What the above considerations show is that it is easy to conceive of an all D-configuration enzyme with the ability to produce D-amino acids from their L-configuration counterparts, given known enzymatic activities. It is therefore accordingly not a difficult evolutionary accomplishment.

And if this is true for the chirally-opposite life-types in spatial proximity, then the condition of true orthogonality breaks down. Inverse Life-2.0 organisms could use Life-1.0 organisms as food sources, and vice versa. It could be not doubted that if it was at all possible for Life-2.0 organisms to make use of resources inadvertently supplied by Life-1.0, they would. Conventional life provides plenty of precedent for this assertion. We could propose a variant of Murphy’s Law: If it is at all physically and evolutionarily possible for one group of organisms to benefit from parasitizing or exploiting another, they will.

All of these considerations assume that Life-2.0 has a similar range of (D-configuration) amino acids as for the L-configuration set for conventional life. Certainly it is logical that the more a postulated ‘weird life’ diverges from known life, the harder it would be for the one to utilize the resources of the other. But on the other hand, if any life-type is presented with a pool of potentially beneficial organic molecules (from a distinct co-existing life-type) over vast time periods, it would seem likely that a way to use them would eventually be found, even if such compounds were initially completely refractory.  (The above Murphy’s Law variant, in action).

And then, if orthogonality of spatially proximal life-types is improbable, it becomes much less likely that the putative Life-2.0 could remain in the shadows. The more interactions, the more likely their presence emerges from the shadows into light.

For the curse of biopolyverse in verse, Inverse Biology :

Would it be excessively perverse?

A stereo-conundrum, or worse?

A whole chiral hop

An L– to D– swap

A biology mirrored, inverse….

References & Details

(In Order of Citation)

 With respect to imbalances in amino acid stereoisomers (‘enantiomeric excesses’) in meteoric sources, see Vandenbussche et al. 2011.

An alternative for spiegel-leben (‘mirror-life’) would be spiegelwesen (‘mirror-being’ / ‘mirror-entity’) which has appeal, but is already used in different German-speaking contexts.

Search for chirally  inverted organisms: “The physicist Paul Davies ……has taken this idea with sufficient seriousness….” Paul Davies has credited the original idea to his wife Paula, as described in the transcript of a radio interview.

‘….and growth was obtained..’ Growth in opposite-chirality nutrient mixes was mentioned in the same interview as above.

‘….the use of D-alanine as a component of peptidoglycan…’ For more information, see Barreteau et al. 2008.

  ‘…D-amino acids have now been found widely distributed among the kingdoms of life…’ Ribosomal synthesis of proteins is restricted to the L-amino acid alphabet, but post-translational modifications of specific peptides to create D-residues at targeted sites is known, initially in invertebrates (Heck et al. 1996) but more recently found also in mammals Torres et al 2006 (platypus venom). Individual D-amino acids are also widely distributed among diverse organisms, and found in plants as well as animals (for example, see Fujitani et al.  2007 ).

‘….Dserine is found in significant amounts in mammalian neural tissue…’. For a review of D-serine in mammalian neurology, see Wolosker et al. 2008.

‘…role in neurotransmission.’ D-serine acts as a ‘co-agonist’ (response modulator) of the receptor for N-methyl- D-aspartate receptor, itself another example of a physiological (modified) D-amino acid. (See Yoshimura & Goto 2008).

This kind of effect has been shown to work with real enzymes.’ Stephen Kent has pioneered the field of total chemical synthesis of small proteins, including D-isomers of enzymes showing reciprocal chiral action (See Milton et al. 1992).

Such mirror-binders have been referred to as spiegelmers….’ For more details, see Nolte et al. 1996.

‘…it would seem likely that a way to use them would eventually be found …’ Note that this wording does NOT indicate any directionality or pre-commitment (teleology) in evolution. An organism which by chance gained an enzymatic means for inefficiently exploiting a new carbon source would gain a selective advantage over its intraspecific competitors, which failed to use the same resource at all. Further selectable incremental improvements in the enzymatic use of the new substrate would follow, until it became a standard option for that microbial group.

*********************************************************************************

Finally, it should be noted that even if no ‘inverse life’ exists on Earth, this does not preclude the possibility of creating it. This is a whole topic for future consideration….

Next posting: Two weeks from now.

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