Structural basis for tRNA methylthiolation by the radical SAM enzyme MiaB.

Numerous post-transcriptional modifications of transfer RNAs have vital roles in translation. The 2-methylthio-N6-isopentenyladenosine (ms2i6A) modification occurs at position 37 (A37) in transfer RNAs that contain adenine in position 36 of the anticodon, and serves to promote efficient A:U codon-anticodon base-pairing and to prevent unintended base pairing by near cognates, thus enhancing translational fidelity1-4. The ms2i6A modification is installed onto isopentenyladenosine (i6A) by MiaB, a radical S-adenosylmethionine (SAM) methylthiotransferase. As a radical SAM protein, MiaB contains one [Fe4S4]RS cluster used in the reductive cleavage of SAM to form a 5'-deoxyadenosyl 5'-radical, which is responsible for removing the C2 hydrogen of the substrate5. MiaB also contains an auxiliary [Fe4S4]aux cluster, which has been implicated6-9 in sulfur transfer to C2 of i6A37. How this transfer takes place is largely unknown. Here we present several structures of MiaB from Bacteroides uniformis. These structures are consistent with a two-step mechanism, in which one molecule of SAM is first used to methylate a bridging µ-sulfido ion of the auxiliary cluster. In the second step, a second SAM molecule is cleaved to a 5'-deoxyadenosyl 5'-radical, which abstracts the C2 hydrogen of the substrate but only after C2 has undergone rehybridization from sp2 to sp3. This work advances our understanding of how enzymes functionalize inert C-H bonds with sulfur.

The ATAC acetyltransferase complex coordinates MAP kinases to regulate JNK target genes.

In response to extracellular cues, signal transduction activates downstream transcription factors like c-Jun to induce expression of target genes. We demonstrate that the ATAC (Ada two A containing) histone acetyltransferase (HAT) complex serves as a transcriptional cofactor for c-Jun at the Jun N-terminal kinase (JNK) target genes Jra and chickadee. ATAC subunits are required for c-Jun occupancy of these genes and for H4K16 acetylation at the Jra enhancer, promoter, and transcribed sequences. Under conditions of osmotic stress, ATAC colocalizes with c-Jun, recruits the upstream kinases Misshapen, MKK4, and JNK, and suppresses further activation of JNK. Relocalization of these MAPKs and suppression of JNK activation by ATAC are dependent on the CG10238 subunit of ATAC. Thus, ATAC governs the transcriptional response to MAP kinase signaling by serving as both a coactivator of transcription and as a suppressor of upstream signaling.

Molecular basis for the bifunctional Uba4-Urm1 sulfur-relay system in tRNA thiolation and ubiquitin-like conjugation.

The chemical modification of tRNA bases by sulfur is crucial to tune translation and to optimize protein synthesis. In eukaryotes, the ubiquitin-related modifier 1 (Urm1) pathway is responsible for the synthesis of 2-thiolated wobble uridine (U34 ). During the key step of the modification cascade, the E1-like activating enzyme ubiquitin-like protein activator 4 (Uba4) first adenylates and thiocarboxylates the C-terminus of its substrate Urm1. Subsequently, activated thiocarboxylated Urm1 (Urm1-COSH) can serve as a sulfur donor for specific tRNA thiolases or participate in ubiquitin-like conjugation reactions. Structural and mechanistic details of Uba4 and Urm1 have remained elusive but are key to understand the evolutionary branch point between ubiquitin-like proteins (UBL) and sulfur-relay systems. Here, we report the crystal structures of full-length Uba4 and its heterodimeric complex with its substrate Urm1. We show how the two domains of Uba4 orchestrate recognition, binding, and thiocarboxylation of the C-terminus of Urm1. Finally, we uncover how the catalytic domains of Uba4 communicate efficiently during the reaction cycle and identify a mechanism that enables Uba4 to protect itself against self-conjugation with its own product, namely activated Urm1-COSH.

Essentiality of the Escherichia coli YgfZ Protein for the In Vivo Thiomethylation of Ribosomal Protein S12 by the RimO Enzyme.

Enzymes carrying Iron-Sulfur (Fe-S) clusters perform many important cellular functions and their biogenesis require complex protein machinery. In mitochondria, the IBA57 protein is essential and promotes assembly of [4Fe-4S] clusters and their insertion into acceptor proteins. YgfZ is the bacterial homologue of IBA57 but its precise role in Fe-S cluster metabolism is uncharacterized. YgfZ is needed for activity of the radical S-adenosyl methionine [4Fe-4S] cluster enzyme MiaB which thiomethylates some tRNAs. The growth of cells lacking YgfZ is compromised especially at low temperature. The RimO enzyme is homologous to MiaB and thiomethylates a conserved aspartic acid in ribosomal protein S12. To quantitate thiomethylation by RimO, we developed a bottom-up LC-MS2 analysis of total cell extracts. We show here that the in vivo activity of RimO is very low in the absence of YgfZ and independent of growth temperature. We discuss these results in relation to the hypotheses relating to the role of the auxiliary 4Fe-4S cluster in the Radical SAM enzymes that make Carbon-Sulfur bonds.

An ancient type of MnmA protein is an iron-sulfur cluster-dependent sulfurtransferase for tRNA anticodons.

Transfer RNA (tRNA) is an adaptor molecule indispensable for assigning amino acids to codons on mRNA during protein synthesis. 2-thiouridine (s2U) derivatives in the anticodons (position 34) of tRNAs for glutamate, glutamine, and lysine are post-transcriptional modifications essential for precise and efficient codon recognition in all organisms. s2U34 is introduced either by (i) bacterial MnmA/eukaryote mitochondrial Mtu1 or (ii) eukaryote cytosolic Ncs6/archaeal NcsA, and the latter enzymes possess iron-sulfur (Fe-S) cluster. Here, we report the identification of novel-type MnmA homologs containing three conserved Cys residues, which could support Fe-S cluster binding and catalysis, in a broad range of bacteria, including thermophiles, Cyanobacteria, Mycobacteria, Actinomyces, Clostridium, and Helicobacter Using EPR spectroscopy, we revealed that Thermus thermophilus MnmA (TtMnmA) contains an oxygen-sensitive [4Fe-4S]-type cluster. Efficient in vitro formation of s2U34 in tRNALys and tRNAGln by holo-TtMnmA occurred only under anaerobic conditions. Mutational analysis of TtMnmA suggested that the Fe-S cluster is coordinated by the three conserved Cys residues (Cys105, Cys108, and Cys200), and is essential for its activity. Evolutionary scenarios for the sulfurtransferases, including the Fe-S cluster containing Ncs6/NcsA s2U thiouridylases and several distantly related sulfurtransferases, are proposed.

Thioredoxin regulates human mercaptopyruvate sulfurtransferase at physiologically-relevant concentrations.

3-Mercaptopyruvate sulfur transferase (MPST) catalyzes the desulfuration of 3-mercaptopyruvate (3-MP) and transfers sulfane sulfur from an enzyme-bound persulfide intermediate to thiophilic acceptors such as thioredoxin and cysteine. Hydrogen sulfide (H2S), a signaling molecule implicated in many physiological processes, can be released from the persulfide product of the MPST reaction. Two splice variants of MPST, differing by 20 amino acids at the N terminus, give rise to the cytosolic MPST1 and mitochondrial MPST2 isoforms. Here, we characterized the poorly-studied MPST1 variant and demonstrated that substitutions in its Ser-His-Asp triad, proposed to serve a general acid-base role, minimally affect catalytic activity. We estimated the 3-MP concentration in murine liver, kidney, and brain tissues, finding that it ranges from 0.4 µmol·kg-1 in brain to 1.4 µmol·kg-1 in kidney. We also show that N-acetylcysteine, a widely-used antioxidant, is a poor substrate for MPST and is unlikely to function as a thiophilic acceptor. Thioredoxin exhibits substrate inhibition, increasing the KM for 3-MP ∼15-fold compared with other sulfur acceptors. Kinetic simulations at physiologically-relevant substrate concentrations predicted that the proportion of sulfur transfer to thioredoxin increases ∼3.5-fold as its concentration decreases from 10 to 1 µm, whereas the total MPST reaction rate increases ∼7-fold. The simulations also predicted that cysteine is a quantitatively-significant sulfane sulfur acceptor, revealing MPST's potential to generate low-molecular-weight persulfides. We conclude that the MPST1 and MPST2 isoforms are kinetically indistinguishable and that thioredoxin modulates the MPST-catalyzed reaction in a physiologically-relevant concentration range.

3-Mercaptopyruvate sulfurtransferase: an enzyme at the crossroads of sulfane sulfur trafficking.

3-Mercaptopyruvate sulfurtransferase (MPST) catalyzes the desulfuration of 3-mercaptopyruvate to generate an enzyme-bound hydropersulfide. Subsequently, MPST transfers the persulfide's outer sulfur atom to proteins or small molecule acceptors. MPST activity is known to be involved in hydrogen sulfide generation, tRNA thiolation, protein urmylation and cyanide detoxification. Tissue-specific changes in MPST expression correlate with ageing and the development of metabolic disease. Deletion and overexpression experiments suggest that MPST contributes to oxidative stress resistance, mitochondrial respiratory function and the regulation of fatty acid metabolism. However, the role and regulation of MPST in the larger physiological context remain to be understood.

Characterization and Reconstitution of Human Lipoyl Synthase (LIAS) Supports ISCA2 and ISCU as Primary Cluster Donors and an Ordered Mechanism of Cluster Assembly.

Lipoyl synthase (LIAS) is an iron-sulfur cluster protein and a member of the radical S-adenosylmethionine (SAM) superfamily that catalyzes the final step of lipoic acid biosynthesis. The enzyme contains two [4Fe-4S] centers (reducing and auxiliary clusters) that promote radical formation and sulfur transfer, respectively. Most information concerning LIAS and its mechanism has been determined from prokaryotic enzymes. Herein, we detail the expression, isolation, and characterization of human LIAS, its reactivity, and evaluation of natural iron-sulfur (Fe-S) cluster reconstitution mechanisms. Cluster donation by a number of possible cluster donor proteins and heterodimeric complexes has been evaluated. [2Fe-2S]-cluster-bound forms of human ISCU and ISCA2 were found capable of reconstituting human LIAS, such that complete product turnover was enabled for LIAS, as monitored via a liquid chromatography-mass spectrometry (LC-MS) assay. Electron paramagnetic resonance (EPR) studies of native LIAS and substituted derivatives that lacked the ability to bind one or the other of LIAS's two [4Fe-4S] clusters revealed a likely order of cluster addition, with the auxiliary cluster preceding the reducing [4Fe-4S] center. These results detail the trafficking of Fe-S clusters in human cells and highlight differences with respect to bacterial LIAS analogs. Likely in vivo Fe-S cluster donors to LIAS are identified, with possible connections to human disease states, and a mechanistic ordering of [4Fe-4S] cluster reconstitution is evident.

In Vitro Production of Ergothioneine Isotopologues.

Ergothioneine is an emerging component of the redox homeostasis system in human cells and in microbial pathogens, such as Mycobacterium tuberculosis and Burkholderia pseudomallei. The synthesis of stable isotope-labeled ergothioneine derivatives may provide important tools for deciphering the distribution, function, and metabolism of this compound in vivo. We describe a general protocol for the production of ergothioneine isotopologues with programmable 2 H, 15 N, 13 C, 34 S, and 33 S isotope labeling patterns. This enzyme-based approach makes efficient use of commercial isotope reagents and is also directly applicable to the synthesis of radio-isotopologues.

Biosynthesis of Chuangxinmycin Featuring a Deubiquitinase-like Sulfurtransferase.

The knowledge on sulfur incorporation mechanism involved in sulfur-containing molecule biosynthesis remains limited. Chuangxinmycin is a sulfur-containing antibiotic with a unique thiopyrano[4,3,2-cd]indole (TPI) skeleton and selective inhibitory activity against bacterial tryptophanyl-tRNA synthetase. Despite the previously reported biosynthetic gene clusters and the recent functional characterization of a P450 enzyme responsible for C-S bond formation, the enzymatic mechanism for sulfur incorporation remains unknown. Here, we resolve this central biosynthetic problem by in vitro biochemical characterization of the key enzymes and reconstitute the TPI skeleton in a one-pot enzymatic reaction. We reveal that the JAMM/MPN+ protein Cxm3 functions as a deubiquitinase-like sulfurtransferase to catalyze a non-classical sulfur-transfer reaction by interacting with the ubiquitin-like sulfur carrier protein Cxm4GG. This finding adds a new mechanism for sulfurtransferase in nature.

Structural and Mechanistic Basis for Anaerobic Ergothioneine Biosynthesis.

Ergothioneine is an emergent factor in cellular redox biochemistry in humans and pathogenic bacteria. Broad consensus has formed around the idea that ergothioneine protects cells against reactive oxygen species. The recent discovery that anaerobic microorganisms make the same metabolite using oxygen-independent chemistry indicates that ergothioneine also plays physiological roles under anoxic conditions. In this report, we describe the crystal structure of the anaerobic ergothioneine biosynthetic enzyme EanB from green sulfur bacterium Chlorobium limicola. This enzyme catalyzes the oxidative sulfurization of N-α-trimethyl histidine. On the basis of structural and kinetic evidence, we describe the catalytic mechanism of this unusual C-S bond-forming reaction. Significant active-site conservation among distant EanB homologues suggests that the oxidative sulfurization of heterocyclic substrates may occur in a broad range of bacteria.

Structural analysis of molybdopterin synthases from two mycobacterial pathogens.

The molybdenum cofactor, composed of molybdopterin and molybdenum, is a necessary compound for the catalytic activity of molybdenum enzymes. Molybdenum cofactor biosynthesis is a conserved multi-step process involving several enzymes. Molybdopterin synthase, a hetero-tetrameric enzyme composed of a pair of MoaE-MoaD subunits, catalyzes the generation of the cis-dithiolene group of molybdopterin in the second step of the process. The cis-dithiolene group can covalently bind molybdenum. Most mycobacterial species possess several genes encoding the full pathway of molybdenum cofactor biosynthesis. In M. smegmatis, the moaD2 and moaE2 genes encode the functional molybdopterin synthase. However, M. tuberculosis has genes encoding several molybdopterin synthase subunit homologs, including moaD1, moaD2, moaE1, moaE2, and moaX, which encodes a MoaD-MoaE fusion protein. Previous studies have shown that moaD2 and moaE2 encode functional molybdopterin synthase. Here, we report the crystal structures of two substrate-free molybdopterin synthases from two different mycobacterial pathogens, M. tuberculosis and M. smegmatis, at 2.1 Šand 2.6 Šresolutions, respectively. The overall structure of both molybdopterin synthases was hetero-tetrameric, consisting of a MoaE2 dimer flanked on either side by single MoaD2 subunits. The carboxyl-terminal domain of MoaD2 inserted into MoaE2, forming the active pocket. A comparison with previously reported molybdopterin synthase structures showed that substrate-binding and catalytic residues were conserved, despite low sequence similarity among these enzymes. The low sequence identity at the MoaE-MoaD heterodimer interface may provide the structural basis to explore mycobacterial inhibitors.

Rhodanese domain-containing sulfurtransferases: multifaceted proteins involved in sulfur trafficking in plants.

Sulfur is an essential element for the growth and development of plants, which synthesize cysteine and methionine from the reductive assimilation of sulfate. Besides its incorporation into proteins, cysteine is the building block for the biosynthesis of numerous sulfur-containing molecules and cofactors. The required sulfur atoms are extracted either directly from cysteine by cysteine desulfurases or indirectly after its catabolic transformation to 3-mercaptopyruvate, a substrate for sulfurtransferases (STRs). Both enzymes are transiently persulfidated in their reaction cycle, i.e. the abstracted sulfur atom is bound to a reactive cysteine residue in the form of a persulfide group. Trans-persulfidation reactions occur when sulfur atoms are transferred to nucleophilic acceptors such as glutathione, proteins, or small metabolites. STRs form a ubiquitous, multigenic protein family. They are characterized by the presence of at least one rhodanese homology domain (Rhd), which usually contains the catalytic, persulfidated cysteine. In this review, we focus on Arabidopsis STRs, presenting the sequence characteristics of all family members as well as their biochemical and structural features. The physiological functions of particular STRs in the biosynthesis of molybdenum cofactor, thio-modification of cytosolic tRNAs, arsenate tolerance, cysteine catabolism, and hydrogen sulfide formation are also discussed.

Structure of the human aminopeptidase XPNPEP3 and comparison of its in vitro activity with Icp55 orthologs: Insights into diverse cellular processes.

The human aminopeptidase XPNPEP3 is associated with cystic kidney disease and TNF-TNFR2 cellular signaling. Its yeast and plant homolog Icp55 processes several imported mitochondrial matrix proteins leading to their stabilization. However, the molecular basis for the diverse roles of these enzymes in the cell is unknown. Here, we report the crystal structure of human XPNPEP3 with bound apstatin product at 1.65 Å resolution, and we compare its in vitro substrate specificity with those of fungal Icp55 enzymes. In contrast to the suggestions by earlier in vivo studies of mitochondrial processing, we found that these enzymes are genuine Xaa-Pro aminopeptidases, which hydrolyze peptides with proline at the second position (P1'). The mitochondrial processing activity involving cleavage of peptides lacking P1' proline was also detected in the purified enzymes. A wide proline pocket as well as molecular complementarity and capping at the S1 substrate site of XPNPEP3 provide the necessary structural features for processing the mitochondrial substrates. However, this activity was found to be significantly lower as compared with Xaa-Pro aminopeptidase activity. Because of similar activity profiles of Icp55 and XPNPEP3, we propose that XPNPEP3 plays the same mitochondrial role in humans as Icp55 does in yeast. Both Xaa-Pro aminopeptidase and mitochondrial processing activities of XPNPEP3 have implications toward mitochondrial fitness and cystic kidney disease. Furthermore, the presence of both these activities in Icp55 elucidates the unexplained processing of the mitochondrial cysteine desulfurase Nfs1 in yeast. The enzymatic and structural analyses reported here provide a valuable molecular framework for understanding the diverse cellular roles of XPNPEP3.

Iron-sulfur cluster biogenesis and trafficking in mitochondria.

The biogenesis of iron-sulfur (Fe/S) proteins in eukaryotes is a multistage, multicompartment process that is essential for a broad range of cellular functions, including genome maintenance, protein translation, energy conversion, and the antiviral response. Genetic and cell biological studies over almost 2 decades have revealed some 30 proteins involved in the synthesis of cellular [2Fe-2S] and [4Fe-4S] clusters and their incorporation into numerous apoproteins. Mechanistic aspects of Fe/S protein biogenesis continue to be elucidated by biochemical and ultrastructural investigations. Here, we review recent developments in the pursuit of constructing a comprehensive model of Fe/S protein assembly in the mitochondrion.

EPR-Derived Structure of a Paramagnetic Intermediate Generated by Biotin Synthase BioB.

Biotin (vitamin B7) is an enzyme cofactor required by organisms from all branches of life but synthesized only in microbes and plants. In the final step of biotin biosynthesis, a radical S-adenosyl-l-methionine (SAM) enzyme, biotin synthase (BioB), converts the substrate dethiobiotin to biotin through the stepwise formation of two C-S bonds. Previous electron paramagnetic resonance (EPR) spectroscopic studies identified a semistable intermediate in the formation of the first C-S bond as 9-mercaptodethiobiotin linked to a paramagnetic [2Fe-2S] cluster through one of its bridging sulfides. Herein, we report orientation-selected pulse EPR spectroscopic results that reveal hyperfine interactions between the [2Fe-2S] cluster and a number of magnetic nuclei (e.g., 57Fe, 15N, 13C, and 2H) introduced in a site-specific manner via biosynthetic methods. Combining these results with quantum chemical modeling gives a structural model of the intermediate showing that C6, the target of the second hydrogen-atom abstraction, is now in close proximity to the nascent thioether sulfur and is ideally positioned for the second C-S bond forming event.

Cleavage of molybdopterin synthase MoaD-MoaE linear fusion by JAMM/MPN+ domain containing metalloprotease DR0402 from Deinococcus radiodurans.

Molybdenum cofactor (Moco), molybdopterin (MPT) complexed with molybdenum, is an essential cofactor required for the catalytic center of diverse enzymes in all domains of life. Since Moco cannot be taken up as a nutrient unlike many other cofactors, Moco requires de novo biosynthesis. During the synthesis of MPT, the sulfur atom on the C-terminus of MoaD is transferred to cyclic pyranopterin monophosphate (cPMP) which is bound in the substrate pocket of MoaE. MoaD is a ubiquitin-like (Ubl) protein and has a C-terminal di-Gly motif which is a common feature of Ubl proteins. Despite the importance of free C terminal di-Gly motif of MoaD as a sulfur carrier, some bacteria encode a fused MPT synthase in which MoaD- and MoaE-like domains are located on a single peptide. Although it has recently been reported that the fused MPT synthase MoaX from Mycobacterium tuberculosis is posttranslationally cleaved into functional MoaD and MoaE in M. smegmatis, the protease responsible for the cleavage of MoaD-MoaE fusion protein has remained unknown to date. Here we report that the JAMM/MPN+ domain containing metalloprotease DR0402 (JAMMDR) from Deinococcus radiodurans can cleave the MoaD-MoaE fusion protein DR2607, the sole MPT synthase in D. radiodurans, generating the MoaD having a C-terminal di-Gly motif. Furthermore, JAMMDR can also cleave off the MoaD from MoaD-eGFP fusion protein suggesting that JAMMDR recognizes the MoaD region rather than MoaE region in the cleaving process of MoaD-MoaE fusion protein.

Molecular basis of function and the unusual antioxidant activity of a cyanobacterial cysteine desulfurase.

Cysteine desulfurases, which supply sulfur for iron-sulfur cluster biogenesis, are broadly distributed in all phyla including cyanobacteria, the progenitors of plant chloroplasts. The SUF (sulfur utilization factor) system is responsible for Fe-S cluster biosynthesis under stress. The suf operon from cyanobacterium Anabaena PCC 7120 showed the presence of a cysteine desulfurase, sufS (alr2495), but not the accessory sulfur-accepting protein (SufE). However, an open reading frame (alr3513) encoding a SufE-like protein (termed AsaE, Anabaena sulfur acceptor E) was found at a location distinct from the suf operon. The purified SufS protein existed as a pyridoxal 5' phosphate (PLP)-containing dimer with a relatively low desulfurase activity. Interestingly, in the presence of the AsaE protein, the catalytic efficiency of this reaction increased 10-fold. In particular, for sulfur mobilization, the AsaE protein partnered only SufS and not other cysteine desulfurases from Anabaena. The SufS protein was found to physically interact with the AsaE protein, demonstrating that AsaE was indeed the missing partner of Anabaena SufS. The conserved cysteine of the SufS or the AsaE protein was essential for activity but not for their physical association. Curiously, overexpression of the SufS protein in Anabaena caused reduced formation of reactive oxygen species on exposure to hydrogen peroxide (H2O2), resulting in superior oxidative stress tolerance to the oxidizing agent when compared with the wild-type strain. Overall, the results highlight the functional interaction between the two proteins that mediate sulfur mobilization, in the cyanobacterial SUF pathway, and further reveal that overexpression of SufS can protect cyanobacteria from oxidative stress.

Biogenesis of reactive sulfur species for signaling by hydrogen sulfide oxidation pathways.

The chemical species involved in H2S signaling remain elusive despite the profound and pleiotropic physiological effects elicited by this molecule. The dominant candidate mechanism for sulfide signaling is persulfidation of target proteins. However, the relatively poor reactivity of H2S toward oxidized thiols, such as disulfides, the low concentration of disulfides in the reducing milieu of the cell and the low steady-state concentration of H2S raise questions about the plausibility of persulfide formation via reaction between an oxidized thiol and a sulfide anion or a reduced thiol and oxidized hydrogen disulfide. In contrast, sulfide oxidation pathways, considered to be primarily mechanisms for disposing of excess sulfide, generate a series of reactive sulfur species, including persulfides, polysulfides and thiosulfate, that could modify target proteins. We posit that sulfide oxidation pathways mediate sulfide signaling and that sulfurtransferases ensure target specificity.

Homology modeling of Homo sapiens lipoic acid synthase: Substrate docking and insights on its binding mode.

Lipoic acid synthase (LIAS) is an iron-sulfur cluster mitochondrial enzyme which catalyzes the final step in the de novo pathway for the biosynthesis of lipoic acid, a potent antioxidant. Recently there has been significant interest in its role in metabolic diseases and its deficiency in LIAS expression has been linked to conditions such as diabetes, atherosclerosis and neonatal-onset epilepsy, suggesting a strong inverse correlation between LIAS reduction and disease status. In this study we use a bioinformatics approach to predict its structure, which would be helpful to understanding its role. A homology model for LIAS protein was generated using X-ray crystallographic structure of Thermosynechococcus elongatus BP-1 (PDB ID: 4U0P). The predicted structure has 93% of the residues in the most favour region of Ramachandran plot. The active site of LIAS protein was mapped and docked with S-Adenosyl Methionine (SAM) using GOLD software. The LIAS-SAM complex was further refined using molecular dynamics simulation within the subsite 1 and subsite 3 of the active site. To the best of our knowledge, this is the first study to report a reliable homology model of LIAS protein. This study will facilitate a better understanding mode of action of the enzyme-substrate complex for future studies in designing drugs that can target LIAS protein.