Rajan Sankaranarayanan's research while affiliated with Centre for Cellular and Molecular Biology and other places

Outliers in scientific observations are often ignored and mostly remain unreported. However, presenting them is always beneficial since they could reflect the actual anomalies that might open new avenues. Here, we describe two examples of the above that came out of the laboratories of two of the pioneers of nucleic acid research in the area of protein biosynthesis, Paul Berg and Donald Crothers. Their work on the identification of D-aminoacyl-tRNA deacylase (DTD) and ‘Discriminator hypothesis’, respectively, were hugely ahead of their time and were partly against the general paradigm at that time. In both of the above works, the smallest and the only achiral amino acid turned out to be an outlier as DTD can act weakly on glycine charged tRNAs with a unique discriminator base of ‘Uracil’. This peculiar nature of glycine remained an enigma for nearly half a century. With a load of available information on the subject by the turn of the century, our work on ‘chiral proofreading’ mechanisms during protein biosynthesis serendipitously led us to revisit these findings. Here, we describe how we uncovered an unexpected connection between them that has implications for evolution of different eukaryotic life forms.

Aldehydes, being an integral part of carbon metabolism, energy generation, and signalling pathways, are ingrained in plant physiology. Land plants have developed intricate metabolic pathways which involve production of reactive aldehydes and its detoxification to survive harsh terrestrial environments. Here, we show that physiologically produced aldehydes, i.e., formaldehyde and methylglyoxal in addition to acetaldehyde, generate adducts with aminoacyl-tRNAs, a substrate for protein synthesis. Plants are unique in possessing two distinct chiral proofreading systems, D-aminoacyl-tRNA deacylase1 (DTD1) and DTD2, of bacterial and archaeal origins, respectively. Extensive biochemical analysis revealed that only archaeal DTD2 can remove the stable D-aminoacyl adducts on tRNA thereby shielding archaea and plants from these system-generated aldehydes. Using Arabidopsis as a model system, we have shown that the loss of DTD2 gene renders plants susceptible to these toxic aldehydes as they generate stable alkyl modification on D-aminoacyl-tRNAs, which are recycled only by DTD2. Bioinformatic analysis identifies the expansion of aldehyde metabolising repertoire in land plant ancestors which strongly correlates with the recruitment of archaeal DTD2. Finally, we demonstrate that the overexpression of DTD2 offers better protection against aldehydes than in wild type Arabidopsis highlighting its role as a multi-aldehyde detoxifier that can be explored as a transgenic crop development strategy.

Aldehydes, being an integral part of carbon metabolism, energy generation and signalling pathways, are ingrained in plant physiology. Land plants have developed intricate metabolic pathways which involve production of reactive aldehydes and its detoxification to survive harsh terrestrial environments. Here, we show that physiologically produced aldehydes i.e., formaldehyde and methylglyoxal in addition to acetaldehyde, generate adducts with aminoacyl-tRNAs, a substrate for protein synthesis. Plants are unique in possessing two distinct chiral proofreading systems, D-aminoacyl-tRNA deacylase1 (DTD1) and DTD2, of bacterial and archaeal origins, respectively. Extensive biochemical analysis revealed that only archaeal DTD2 can remove the stable D-aminoacyl adducts on tRNA thereby shielding archaea and plants from these system-generated aldehydes. Using Arabidopsis as a model system, we have shown that the loss of DTD2 gene renders plants susceptible to these toxic aldehydes as they generate stable alkyl modification on D-aminoacyl-tRNAs, which are recycled only by DTD2. Bioinformatic analysis identifies the expansion of aldehyde metabolising repertoire in land plant ancestors which strongly correlates with the recruitment of archaeal DTD2. Finally, we demonstrate that the overexpression of DTD2 offers better protection against aldehydes than in wild-type Arabidopsis highlighting its role as a multi-aldehyde detoxifier that can be explored as a transgenic crop development strategy.

Land plants have developed intricate metabolic pathways which involve production of reactive aldehydes and its detoxification to survive harsh terrestrial environments. Aldehydes, being an integral part of carbon metabolism, energy generation and signalling pathways, are ingrained in plant physiology. Here, we show that physiologically abundantly produced aldehydes i.e., formaldehyde and methylglyoxal in addition to acetaldehyde, generate adducts with aminoacyl-tRNAs, a substrate for protein synthesis. Plants are unique in possessing two distinct chiral proofreading systems, D-aminoacyl-tRNA deacylase1 (DTD1) and DTD2, of bacterial and archaeal origins, respectively. Extensive biochemical analysis revealed that only archaeal DTD2 can remove the stable D-aminoacyl adducts on tRNA thereby shielding archaea and plants from these system-generated aldehydes. Using Arabidopsis as a model system, we have shown that the loss of DTD2 gene renders plants susceptible to these toxic aldehydes as they generate stable alkyl modification on D-aminoacyl-tRNAs, which are recycled only by DTD2. Bioinformatic analysis identifies the expansion of aldehyde metabolising repertoire in land plant ancestors which strongly correlates with the recruitment of archaeal DTD2. Finally, we demonstrate that the overexpression of DTD2 offers better protection against aldehydes than in wild-type Arabidopsis highlighting its role as a multi-aldehyde detoxifier that can be used as a transgenic crop development strategy.

Land plants have developed intricate metabolic pathways which involve production of reactive aldehydes and its detoxification to survive harsh terrestrial environments. Aldehydes, being an integral part of carbon metabolism, energy generation and signalling pathways, are ingrained in plant physiology. Here, we show that physiologically abundantly produced aldehydes i.e., formaldehyde and methylglyoxal in addition to acetaldehyde, generate adducts with aminoacyl-tRNAs, a substrate for protein synthesis. Plants are unique in possessing two distinct chiral proofreading systems, D-aminoacyl-tRNA deacylase1 (DTD1) and DTD2, of bacterial and archaeal origins, respectively. Extensive biochemical analysis revealed that only archaeal DTD2 can remove the stable D-aminoacyl adducts on tRNA thereby shielding archaea and plants from these system-generated aldehydes. Using Arabidopsis as a model system, we have shown that the loss of DTD2 gene renders plants susceptible to these toxic aldehydes as they generate stable alkyl modification on D-aminoacyl-tRNAs, which are recycled only by DTD2. Bioinformatic analysis identifies the expansion of aldehyde metabolising repertoire in land plant ancestors which strongly correlates with the recruitment of archaeal DTD2. Finally, we demonstrate that the overexpression of DTD2 offers better protection against aldehydes than in wild-type Arabidopsis highlighting its role as a multi-aldehyde detoxifier that can be used as a transgenic crop development strategy.

Land plants have developed intricate metabolic pathways which involve production of reactive aldehydes and its detoxification to survive harsh terrestrial environments. Aldehydes, being an integral part of carbon metabolism, energy generation and signalling pathways, are ingrained in plant physiology. Here, we show that physiologically abundantly produced aldehydes i.e., formaldehyde and methylglyoxal in addition to acetaldehyde, generate adducts with aminoacyl-tRNAs, a substrate for protein synthesis. Plants are unique in possessing two distinct chiral proofreading systems, D-aminoacyl-tRNA deacylase1 (DTD1) and DTD2, of bacterial and archaeal origins, respectively. Extensive biochemical analysis revealed that only archaeal DTD2 can remove the stable D-aminoacyl adducts on tRNA thereby shielding archaea and plants from these system-generated aldehydes. Using Arabidopsis as a model system, we have shown that the loss of DTD2 gene renders plants susceptible to these toxic aldehydes as they generate stable alkyl modification on D-aminoacyl-tRNAs, which are recycled only by DTD2. Bioinformatic analysis identifies the expansion of aldehyde metabolising repertoire in land plant ancestors which strongly correlates with the recruitment of archaeal DTD2. Finally, we demonstrate that the overexpression of DTD2 offers better protection against aldehydes than in wild-type Arabidopsis highlighting its role as a multi-aldehyde detoxifier that can be used as a transgenic crop development strategy.

Fatty acids are used in fundamental cellular processes, such as membrane biogenesis, energy generation, post-translational modification of proteins, and so forth. These processes require the activation of fatty acids by adenosine triphosphate (ATP), followed by condensation with coenzyme-A (CoA), catalyzed by the omnipresent enzyme called Fatty acyl-CoA ligases (FACLs). However, Fatty acyl-AMP ligases (FAALs), the structural homologs of FACLs, operate in an unprecedented CoA-independent manner. FAALs transfer fatty acids to the acyl carrier protein (ACP) domain of polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) for the biosynthesis of various antibiotics, lipopeptides, virulent complex lipids, and so forth in bacteria. Recent structural and biochemical insights from our group provide a detailed understanding of the mode of CoA rejection and ACP acceptance by FAALs. In this review, we have discussed advances in the mechanistic, evolutionary, and functional understanding of FAALs and FAAL-like domains across life forms. Here, we are proposing a "Five-tier" mechanistic model to explain the specificity of FAALs. We further demonstrate how FAAL-like domains have been repurposed into a new family of proteins in eukaryotes with a novel function in lipid metabolism.

Plants have two endosymbiotic organelles originated from two bacterial ancestors. The transition from an independent bacterium to a successful organelle would have required extensive rewiring of biochemical networks for its integration with archaeal host. Here, using Arabidopsis as a model system, we show that plant D-aminoacyl-tRNA deacylase 1 (DTD1), of bacterial origin, is detrimental to organellar protein synthesis owing to its changed tRNA recognition code. Plants survive this conflict by spatially restricting the conflicted DTD1 to the cytosol. In addition, plants have targeted archaeal DTD2 to both the organelles as it is compatible with their translation machinery due to its strict D-chiral specificity and lack of tRNA determinants. Intriguingly, plants have confined bacterial-derived DTD1 to work in archaeal-derived cytosolic compartment whereas archaeal DTD2 is targeted to bacterial-derived organelles. Overall, the study provides a remarkable example of the criticality of optimization of biochemical networks for survival and evolution of plant mitochondria and chloroplast.

Homochirality of the cellular proteome is attributed to the L-chiral bias of the translation apparatus. The chiral specificity of enzymes was elegantly explained using the 'four-location' model by Koshland two decades ago. In accordance with the model, it was envisaged and noted that some aminoacyl-tRNA synthetases (aaRS) that charge larger amino acids are porous to D-amino acids. However, a recent study showed that alanyl-tRNA synthetase (AlaRS) can mischarge D-alanine and that its editing domain, but not the universally present D-aminoacyl-tRNA deacylase (DTD), is responsible for correcting the chirality-based error. Here, using in vitro and in vivo data coupled with structural analysis, we show that AlaRS catalytic site is a strict D-chiral rejection system and therefore does not activate D-alanine. It obviates the need for AlaRS editing domain to be active against D-Ala-tRNAAla and we show that it is indeed the case as it only corrects L-serine and glycine mischarging. We further provide direct biochemical evidence showing activity of DTD on smaller D-aa-tRNAs that corroborates with the L-chiral rejection mode of action proposed earlier. Overall, while removing anomalies in the fundamental recognition mechanisms, the current study further substantiates how chiral fidelity is perpetuated during protein biosynthesis.