Redox‐dependent lipoylation of mitochondrial proteins in Plasmodium falciparum

Lipoate scavenging from the human host is essential for malaria parasite survival. Scavenged lipoate is covalently attached to three parasite proteins: the H‐protein and the E2 subunits of branched chain amino acid dehydrogenase (BCDH) and α‐ketoglutarate dehydrogenase (KDH). We show mitochondrial localization for the E2 subunits of BCDH and KDH, similar to previously localized H‐protein, demonstrating that all three lipoylated proteins reside in the parasite mitochondrion. The lipoate ligase 1, LipL1, has been shown to reside in the mitochondrion and it catalyses the lipoylation of the H‐protein; however, we show that LipL1 alone cannot lipoylate BCDH or KDH. A second mitochondrial protein with homology to lipoate ligases, LipL2, does not show ligase activity and is not capable of lipoylating any of the mitochondrial substrates. Instead, BCDH and KDH are lipoylated through a novel mechanism requiring both LipL1 and LipL2. This mechanism is sensitive to redox conditions where BCDH and KDH are exclusively lipoylated under strong reducing conditions in contrast to the H‐protein which is preferentially lipoylated under less reducing conditions. Thus, malaria parasites contain two different routes of mitochondrial lipoylation, an arrangement that has not been described for any other organism.     

Unravelling the lipoyl‐relay of exogenous lipoate utilization in Bacillus subtilis

Lipoate is an essential cofactor for key enzymes of oxidative and one‐carbon metabolism. It is covalently attached to E2 subunits of dehydrogenase complexes and GcvH, the H subunit of the glycine cleavage system. Bacillus subtilis possess two protein lipoylation pathways: biosynthesis and scavenging. The former requires octanoylation of GcvH, insertion of sulfur atoms and amidotransfer of the lipoate to E2s, catalyzed by LipL. Lipoate scavenging is mediated by a lipoyl protein ligase (LplJ) that catalyzes a classical two‐step ATP‐dependent reaction. Although these pathways were thought to be redundant, a ?lipL mutant, in which the endogenous lipoylation pathway of E2 subunits is blocked, showed growth defects in minimal media even when supplemented with lipoate and despite the presence of a functional LplJ. In this study, we demonstrate that LipL is essential to modify E2 subunits of branched chain ketoacid and pyruvate dehydrogenases during lipoate scavenging. The crucial role of LipL during lipoate utilization relies on the strict substrate specificity of LplJ, determined by charge complementarity between the ligase and the lipoylable subunits. This new lipoyl‐relay required for lipoate scavenging highlights the relevance of the amidotransferase as a valid target for the design of new antimicrobial agents among Gram‐positive pathogens.     

A novel amidotransferase required for lipoic acid cofactor assembly in Bacillus subtilis

In the companion paper we reported that Bacillus subtilis requires three proteins for lipoic acid metabolism, all of which are members of the lipoate protein ligase family. Two of the proteins, LipM and LplJ, have been shown to be an octanoyltransferase and a lipoate : protein ligase respectively. The third protein, LipL, is essential for lipoic acid synthesis, but had no detectable octanoyltransferase or ligase activity either in vitro or in vivo. We report that LipM specifically modifies the glycine cleavage system protein, GcvH, and therefore another mechanism must exist for modification of other lipoic acid requiring enzymes (e.g. pyruvate dehydrogenase). We show that this function is provided by LipL, which catalyses the amidotransfer (transamidation) of the octanoyl moiety from octanoyl‐GcvH to the E2 subunit of pyruvate dehydrogenase. LipL activity was demonstrated in vitro with purified components and proceeds via a thioester‐linked acyl‐enzyme intermediate. As predicted, ΔgcvH strains are lipoate auxotrophs. LipL represents a new enzyme activity. It is a GcvH:[lipoyl domain] amidotransferase that probably uses a Cys‐Lys catalytic dyad. Although the active site cysteine residues of LipL and LipB are located in different positions within the polypeptide chains, alignment of their structures show these residues occupy similar positions. Thus, these two homologous enzymes have convergent architectures.     

Scavenging of the cofactor lipoate is essential for the survival of the malaria parasite Plasmodium falciparum

Lipoate is an essential cofactor for key enzymes of oxidative metabolism. Plasmodium falciparum possesses genes for lipoate biosynthesis and scavenging, but it is not known if these pathways are functional, nor what their relative contribution to the survival of intraerythrocytic parasites might be. We detected in parasite extracts four lipoylated proteins, one of which cross-reacted with antibodies against the E2 subunit of apicoplast-localized pyruvate dehydrogenase (PDH). Two highly divergent parasite lipoate ligase A homologues (LplA), LipL1 (previously identified as LplA) and LipL2, restored lipoate scavenging in lipoylation-deficient bacteria, indicating that Plasmodium has functional lipoate-scavenging enzymes. Accordingly, intraerythrocytic parasites scavenged radiolabelled lipoate and incorporated it into three proteins likely to be mitochondrial. Scavenged lipoate was not attached to the PDH E2 subunit, implying that lipoate scavenging drives mitochondrial lipoylation, while apicoplast lipoylation relies on biosynthesis. The lipoate analogue 8-bromo-octanoate inhibited LipL1 activity and arrested P. falciparum in vitro growth, decreasing the incorporation of radiolabelled lipoate into parasite proteins. Furthermore, growth inhibition was prevented by lipoate addition in the medium. These results are consistent with 8-bromo-octanoate specifically interfering with lipoate scavenging. Our study suggests that lipoate metabolic pathways are not redundant, and that lipoate scavenging is critical for Plasmodium intraerythrocytic survival.     

Activation and competition of lipoylation of H protein and its hydrolysis in a reaction cascade catalyzed by the multifunctional enzyme lipoate–protein ligase A

Protein lipoylation is essential for the function of many key enzymes but barely studied kinetically. Here, the two-step reaction cascade of H protein lipoylation catalyzed by the multifunctional enzyme lipoate–protein ligase A (LplA) was quantitatively and differentially studied. We discovered new phenomena and unusual kinetics of the cascade: (a) the speed of the first reaction is faster than the second one by two orders of magnitude, leading to high accumulation of the intermediate lipoyl-AMP (Lip-AMP); (b) Lip-AMP is hydrolyzed, but only significantly at the presence of H protein and in competition with the lipoylation; (c) both the lipoylation of H protein and its hydrolysis is enhanced by the apo and lipoylated forms of H protein and a mutant without the lipoylation site. A conceptual mechanistic model is proposed to explain these experimental observations in which conformational change of LplA upon interaction with H protein and competitive nucleophilic attacks play key roles.     

Interplay between Yersinia pestis and its flea vector in lipoate metabolism

To thrive, vector-borne pathogens must survive in the vector’s gut. How these pathogens successfully exploit this environment in time and space has not been extensively characterized. Using Yersinia pestis (the plague bacillus) and its flea vector, we developed a bioluminescence-based approach and employed it to investigate the mechanisms of pathogenesis at an unprecedented level of detail. Remarkably, lipoylation of metabolic enzymes, via the biosynthesis and salvage of lipoate, increases the Y. pestis transmission rate by fleas. Interestingly, the salvage pathway’s lipoate/octanoate ligase LplA enhances the first step in lipoate biosynthesis during foregut colonization but not during midgut colonization. Lastly, Y. pestis primarily uses lipoate provided by digestive proteolysis (presumably as lipoyl peptides) rather than free lipoate in blood, which is quickly depleted by the vector. Thus, spatial and temporal factors dictate the bacterium’s lipoylation strategies during an infection, and replenishment of lipoate by digestive proteolysis in the vector might constitute an Achilles’ heel that is exploited by pathogens.Subject terms: Bacterial pathogenesis, Metabolism     

Apicoplast lipoic acid protein ligase B is not essential for Plasmodium falciparum

Lipoic acid (LA) is an essential cofactor of alpha-keto acid dehydrogenase complexes (KADHs) and the glycine cleavage system. In Plasmodium, LA is attached to the KADHs by organelle-specific lipoylation pathways. Biosynthesis of LA exclusively occurs in the apicoplast, comprising octanoyl-[acyl carrier protein]: protein N-octanoyltransferase (LipB) and LA synthase. Salvage of LA is mitochondrial and scavenged LA is ligated to the KADHs by LA protein ligase 1 (LplA1). Both pathways are entirely independent, suggesting that both are likely to be essential for parasite survival. However, disruption of the LipB gene did not negatively affect parasite growth despite a drastic loss of LA (>90%). Surprisingly, the sole, apicoplast-located pyruvate dehydrogenase still showed lipoylation, suggesting that an alternative lipoylation pathway exists in this organelle. We provide evidence that this residual lipoylation is attributable to the dual targeted, functional lipoate protein ligase 2 (LplA2). Localisation studies show that LplA2 is present in both mitochondrion and apicoplast suggesting redundancy between the lipoic acid protein ligases in the erythrocytic stages of P. falciparum.     

Lipoate protein ligase B primarily recognizes the C8-phosphopantetheine arm of its donor substrate and weakly binds the acyl carrier protein

Lipoic acid is a sulfur-containing cofactor indispensable for the function of several metabolic enzymes. In microorganisms, lipoic acid can be salvaged from the surroundings by lipoate protein ligase A (LplA), an ATP-dependent enzyme. Alternatively, it can be synthesized by the sequential actions of lipoate protein ligase B (LipB) and lipoyl synthase (LipA). LipB takes up the octanoyl chain from C₈-acyl carrier protein (C₈-ACP), a byproduct of the type II fatty acid synthesis pathway, and transfers it to a conserved lysine of the lipoyl domain of a dehydrogenase. However, the molecular basis of its substrate recognition is still not fully understood. Using Escherichia coli LipB as a model enzyme, we show here that the octanoyl-transferase mainly recognizes the 4′-phosphopantetheine-tethered acyl-chain of its donor substrate and weakly binds the apo-acyl carrier protein. We demonstrate LipB can accept octanoate from its own ACP and noncognate ACPs, as well as C₈-CoA. Furthermore, our ¹H saturation transfer difference and ³¹P NMR studies demonstrate the binding of adenosine, as well as the phosphopantetheine arm of CoA to LipB, akin to binding to LplA. Finally, we show a conserved ⁷¹RGG⁷³ loop, analogous to the lipoate-binding loop of LplA, is required for full LipB activity. Collectively, our studies highlight commonalities between LipB and LplA in their mechanism of substrate recognition. This knowledge could be of significance in the treatment of mitochondrial fatty acid synthesis related disorders.     

Comparative Genomic Analysis Reveals 2-Oxoacid Dehydrogenase Complex Lipoylation Correlation with Aerobiosis in Archaea

Metagenomic analyses have advanced our understanding of ecological microbial diversity, but to what extent can metagenomic data be used to predict the metabolic capacity of difficult-to-study organisms and their abiotic environmental interactions? We tackle this question, using a comparative genomic approach, by considering the molecular basis of aerobiosis within archaea. Lipoylation, the covalent attachment of lipoic acid to 2-oxoacid dehydrogenase multienzyme complexes (OADHCs), is essential for metabolism in aerobic bacteria and eukarya. Lipoylation is catalysed either by lipoate protein ligase (LplA), which in archaea is typically encoded by two genes (LplA-N and LplA-C), or by a lipoyl(octanoyl) transferase (LipB or LipM) plus a lipoic acid synthetase (LipA). Does the genomic presence of lipoylation and OADHC genes across archaea from diverse habitats correlate with aerobiosis? First, analyses of 11,826 biotin protein ligase (BPL)-LplA-LipB transferase family members and 147 archaeal genomes identified 85 species with lipoylation capabilities and provided support for multiple ancestral acquisitions of lipoylation pathways during archaeal evolution. Second, with the exception of the Sulfolobales order, the majority of species possessing lipoylation systems exclusively retain LplA, or either LipB or LipM, consistent with archaeal genome streamlining. Third, obligate anaerobic archaea display widespread loss of lipoylation and OADHC genes. Conversely, a high level of correspondence is observed between aerobiosis and the presence of LplA/LipB/LipM, LipA and OADHC E2, consistent with the role of lipoylation in aerobic metabolism. This correspondence between OADHC lipoylation capacity and aerobiosis indicates that genomic pathway profiling in archaea is informative and that well characterized pathways may be predictive in relation to abiotic conditions in difficult-to-study extremophiles. Given the highly variable retention of gene repertoires across the archaea, the extension of comparative genomic pathway profiling to broader metabolic and homeostasis networks should be useful in revealing characteristics from metagenomic datasets related to adaptations to diverse environments.     

A phosphopantetheinyl transferase that is essential for mitochondrial fatty acid biosynthesis

In this study we report the molecular genetic characterization of the Arabidopsis mitochondrial phosphopantetheinyl transferase (mtPPT), which catalyzes the phosphopantetheinylation and thus activation of mitochondrial acyl carrier protein (mtACP) of mitochondrial fatty acid synthase (mtFAS). This catalytic capability of the purified mtPPT protein (encoded by AT3G11470) was directly demonstrated in an in vitro assay that phosphopantetheinylated mature Arabidopsis apo‐mtACP isoforms. The mitochondrial localization of the AT3G11470‐encoded proteins was validated by the ability of their N‐terminal 80‐residue leader sequence to guide a chimeric GFP protein to this organelle. A T‐DNA‐tagged null mutant mtppt‐1 allele shows an embryo‐lethal phenotype, illustrating a crucial role of mtPPT for embryogenesis. Arabidopsis RNAi transgenic lines with reduced mtPPT expression display typical phenotypes associated with a deficiency in the mtFAS system, namely miniaturized plant morphology, slow growth, reduced lipoylation of mitochondrial proteins, and the hyperaccumulation of photorespiratory intermediates, glycine and glycolate. These morphological and metabolic alterations are reversed when these plants are grown in a non‐photorespiratory condition (i.e. 1% CO₂ atmosphere), demonstrating that they are a consequence of a deficiency in photorespiration due to the reduced lipoylation of the photorespiratory glycine decarboxylase.     

Solution NMR characterization of apical membrane antigen 1 and small molecule interactions as a basis for designing new antimalarials

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) plays an important role in the invasion by merozoites of human red blood cells during a malaria infection. A key region of PfAMA1 is a conserved hydrophobic cleft formed by 12 hydrophobic residues. As anti‐apical membrane antigen 1 antibodies and other inhibitory molecules that target this hydrophobic cleft are able to block the invasion process, PfAMA1 is an attractive target for the development of strain‐transcending antimalarial agents. As solution nuclear magnetic resonance spectroscopy is a valuable technique for the rapid characterization of protein–ligand interactions, we have determined the sequence‐specific backbone assignments for PfAMA1 from two P. falciparum strains, FVO and 3D7. Both selective labelling and unlabelling strategies were used to complement triple‐resonance experiments in order to facilitate the assignment process. We have then used these assignments for mapping the binding sites for small molecules, including benzimidazoles, pyrazoles and 2‐aminothiazoles, which were selected on the basis of their affinities measured from surface plasmon resonance binding experiments. Among the compounds tested, benzimidazoles showed binding to a similar region on both FVO and 3D7 PfAMA1, suggesting that these compounds are promising scaffolds for the development of novel PfAMA1 inhibitors. Copyright © 2016 John Wiley & Sons, Ltd.     

A novel two-gene requirement for the octanoyltransfer reaction of Bacillus subtilis lipoic acid biosynthesis

The Bacillus subtilis genome encodes three apparent lipoyl ligase homologues: yhfJ, yqhM and ywfL, which we have renamed lplJ, lipM and lipL respectively. We show that LplJ encodes the sole lipoyl ligase of this bacterium. Physiological and biochemical characterization of a ΔlipM strain showed that LipM is absolutely required for the endogenous lipoylation of all lipoate-dependent proteins, confirming its role as the B. subtilis octanoyltransferase. However, we also report that in contrast to Escherichia coli, B. subtilis requires a third protein for lipoic acid assembly, LipL. B. subtilis ΔlipL strains are unable to synthesize lipoic acid despite the presence of LipM and the sulphur insertion enzyme, LipA, which should suffice for lipoic acid biosynthesis based on the E. coli model. LipM is only required for the endogenous lipoylation pathway, whereas LipL also plays a role in lipoic acid scavenging. Expression of E. coli lipB allows growth of B. subtilisΔlipL or ΔlipM strains in the absence of supplements. In contrast, growth of an E. coliΔlipB strain can be complemented with lipM, but not lipL. These data together with those of the companion article provide evidence that LipM and LipL catalyse sequential reactions in a novel pathway for lipoic acid biosynthesis.     

CNI1/ATL31, a RING‐type ubiquitin ligase that functions in the carbon/nitrogen response for growth phase transition in Arabidopsis seedlings

Plants are able to sense and respond to changes in the balance between carbon (C) and nitrogen (N) metabolite availability, known as the C/N response. During the transition to photoautotrophic growth following germination, growth of seedlings is arrested if a high external C/N ratio is detected. To clarify the mechanisms for C/N sensing and signaling during this transition period, we screened a large collection of FOX transgenic plants, overexpressing full‐length cDNAs, for individuals able to continue post‐germinative growth under severe C/N stress. One line, cni1‐D (carbon/nitrogen insensitive 1‐dominant), was shown to have a suppressed sensitivity to C/N conditions at both the physiological and molecular level. The CNI1 cDNA encoded a predicted RING‐type ubiquitin ligase previously annotated as ATL31. Overexpression of ATL31 was confirmed to be responsible for the cni1‐D phenotype, and a knock‐out of this gene resulted in hypersensitivity to C/N conditions during post‐germinative growth. The ATL31 protein was confirmed to contain ubiquitin ligase activity using an in vitro assay system. Moreover, removal of this ubiquitin ligase activity from the overexpressed protein resulted in the loss of the mutant phenotype. Taken together, these data demonstrated that CNI1/ATL31 activity is required for the plant C/N response during seedling growth transition.     

X‐ray crystal structures of an orally available aminopeptidase inhibitor,Tosedostat, bound to anti‐malarial drug targets PfA‐M1 and PfA‐M17

New anti‐malarial treatments are desperately required to face the spread of drug resistant parasites. Inhibition of metalloaminopeptidases, PfA‐M1 and PfA‐M17, is a validated therapeutic strategy for treatment of Plasmodium falciparum malaria. Here, we describe the crystal structures of PfA‐M1 and PfA‐M17 bound to chemotherapeutic agent Tosedostat. The inhibitor occupies the enzymes' putative product egress channels in addition to the substrate binding pockets; however, adopts different binding poses when bound to PfA‐M1 and PfA‐M17. These findings will be valuable for the continued development of selective inhibitors of PfA‐M1 and PfA‐M17. Proteins 2015; 83:789–795. © 2015 Wiley Periodicals, Inc.     

Detailed functional characterization of glycosylated and nonglycosylated variants of malaria vaccine candidate PfAMA1 produced in Nicotiana benthamiana and analysis of growth inhibitory responses in rabbits

One of the most promising malaria vaccine candidate antigens is the Plasmodium falciparum apical membrane antigen 1 (PfAMA1). Several studies have shown that this blood‐stage antigen can induce strong parasite growth inhibitory antibody responses. PfAMA1 contains up to six recognition sites for N‐linked glycosylation, a post‐translational modification that is absent in P. falciparum. To prevent any potential negative impact of N‐glycosylation, the recognition sites have been knocked out in most PfAMA1 variants expressed in eukaryotic hosts. However, N‐linked glycosylation may increase efficacy by improving immunogenicity and/or focusing the response towards relevant epitopes by glycan masking. We describe the production of glycosylated and nonglycosylated PfAMA1 in Nicotiana benthamiana and its detailed characterization in terms of yield, integrity and protective efficacy. Both PfAMA1 variants accumulated to high levels (>510 μg/g fresh leaf weight) after transient expression, and high‐mannose‐type N‐glycans were confirmed for the glycosylated variant. No significant differences between the N. benthamiana and Pichia pastoris PfAMA1 variants were detected in conformation‐sensitive ligand‐binding studies. Specific titres of >2 × 10⁶ were induced in rabbits, and strong reactivity with P. falciparum schizonts was observed in immunofluorescence assays, as well as up to 100% parasite growth inhibition for both variants, with IC₅₀ values of ~35 μg/mL. Competition assays indicated that a number of epitopes were shielded from immune recognition by N‐glycans, warranting further studies to determine how glycosylation can be used for the directed targeting of immune responses. These results highlight the potential of plant transient expression systems as a production platform for vaccine candidates.     

An NAD synthetic reaction bypasses the lipoate requirement for aerobic growth of Escherichia coli strains blocked in succinate catabolism

The lipoate coenzyme is essential for function of the pyruvate (PDH) and 2‐oxoglutarate (OGDH) dehydrogenases and thus for aerobic growth of Escherichia coli. LipB catalyzes the first step in lipoate synthesis, transfer of an octanoyl moiety from the fatty acid synthetic intermediate, octanoyl‐ACP, to PDH and OGDH. E. coli also encodes LplA, a ligase that in presence of exogenous octanoate (or lipoate) can bypass loss of LipB. LplA imparts ΔlipB strains with a ‘leaky’ growth phenotype on aerobic glucose minimal medium supplemented with succinate (which bypasses the OGDH‐catalyzed reaction), because it scavenges an endogenous octanoate pool to activate PDH. Here we characterize a ΔlipB suppressor strain that did not require succinate supplementation, but did require succinyl‐CoA ligase, confirming the presence of alternative source(s) of cytosolic succinate. We report that suppression requires inactivation of succinate dehydrogenase (SDH), which greatly reduces the cellular requirement for succinate. In the suppressor strain succinate is produced by three enzymes, any one of which will suffice in the absence of SDH. These three enzymes are: trace levels of OGDH, the isocitrate lyase of the glyoxylate shunt and an unanticipated source, aspartate oxidase, the enzyme catalyzing the first step of nicotinamide biosynthesis.     

A novel tomato SUMO E3 ligase,SlSIZ1, confers drought tolerance in transgenic tobacco

SUMOylation is an important post‐translational modification process that regulates different cellular functions in eukaryotes. SIZ/PIAS‐type SAP and Miz1 (SIZ1) proteins exhibit SUMO E3 ligase activity, which modulates SUMOylation. However, SIZ1 in tomato has been rarely investigated. In this study, a tomato SIZ1 gene (SlSIZ1) was isolated and its molecular characteristics and role in tolerance to drought stress are described. SlSIZ1 was up‐regulated by cold, sodium chloride (NaCl), polyethylene glycol (PEG), hydrogen peroxide (H₂O₂) and abscisic acid (ABA), and the corresponding proteins were localized in the nucleus. The expression of SlSIZ1 in Arabidopsis thaliana (Arabidopsis) siz1‐2 mutants partially complemented the phenotypes of dwarf, cold sensitivity and ABA hypersensitivity. SlSIZ1 also exhibited the activity of SUMO E3 ligase to promote the accumulation of SUMO conjugates. Under drought stress, the ectopic expression of SlSIZ1 in transgenic tobacco lines enhanced seed germination and reduced the accumulation of reactive oxygen species. SlSIZ1 overexpression conferred the plants with improved growth, high free proline content, minimal malondialdehyde accumulation and increased accumulation of SUMO conjugates. SlSIZ1 is a functional homolog of Arabidopsis SIZ1 with SUMO E3 ligase activity. Therefore, overexpression of SlSIZ1 enhanced the tolerance of transgenic tobacco to drought stress.     

The Streptomyces coelicolor Lipoate-protein Ligase Is a Circularly Permuted Version of the Escherichia coli Enzyme Composed of Discrete Interacting Domains

Lipoate-protein ligases are used to scavenge lipoic acid from the environment and attach the coenzyme to its cognate proteins, which are generally the E2 components of the 2-oxoacid dehydrogenases. The enzymes use ATP to activate lipoate to its adenylate, lipoyl-AMP, which remains tightly bound in the active site. This mixed anhydride is attacked by the ϵ-amino group of a specific lysine present on a highly conserved acceptor protein domain, resulting in the amide-linked coenzyme. The Streptomyces coelicolor genome encodes only a single putative lipoate ligase. However, this protein had only low sequence identity (<25%) to the lipoate ligases of demonstrated activity and appears to be a circularly permuted version of the known lipoate ligase proteins in that the canonical C-terminal domain seems to have been transposed to the N terminus. We tested the activity of this protein both by in vivo complementation of an Escherichia coli ligase-deficient strain and by in vitro assays. Moreover, when the domains were rearranged into a protein that mimicked the arrangement found in the canonical lipoate ligases, the enzyme retained complementation activity. Finally, when the two domains were separated into two proteins, both domain-containing proteins were required for complementation and catalysis of the overall ligase reaction in vitro. However, only the large domain-containing protein was required for transfer of lipoate from the lipoyl-AMP intermediate to the acceptor proteins, whereas both domain-containing proteins were required to form lipoyl-AMP.     

Recognition of gluconeogenic enzymes; Icl1, Fbp1, and Mdh2 by Gid4 ligase: A molecular docking study

The pro/N‐degron pathway is an evolved protein degradation pathway through the ubiquitin‐proteasome system. It is a vital pathway to attain protein homeostasis inside the liver cells with varying glucose levels. N‐terminal proline exists in more than 300 proteins in Saccharomyces cerevisiae, but only three of them are the gluconeogenic enzymes; isocitrate lyase (Icl1), fructose‐1,6‐bisphosphatase (Fbp1), and malate dehydrogenase (Mdh2). The present in silico study aims to structurally illustrate the binding of Icl1 enzyme to Gid4 ligase concerning its peers; Fbp1 and Mdh2. Based on the molecular docking scores and interactions, one can attribute the binding stability of Gid4 with degrons, to peptides of length six up to eight from the N‐terminal. Moreover, the percent change in the docking score provides a rationale for the unique Gid4‐Icl1^1‐4 interaction. The present study provides insights on the binding attitude of Gid4 ligase to degrons of different lengths, so one will consider in designing peptidomimetics to target Gid4 ligase.