Toward the mechanism of eIF4F-mediated ribosomal attachment to mammalian capped mRNAs.

Ribosomal attachment to mammalian capped mRNAs is achieved through the cap-eukaryotic initiation factor 4E (eIF4E)-eIF4G-eIF3-40S chain of interactions, but the mechanism by which mRNA enters the mRNA-binding channel of the 40S subunit remains unknown. To investigate this process, we recapitulated initiation on capped mRNAs in vitro using a reconstituted translation system. Formation of initiation complexes at 5'-terminal AUGs was stimulated by the eIF4E-cap interaction and followed "the first AUG" rule, indicating that it did not occur by backward scanning. Initiation complexes formed even at the very 5' end of mRNA, implying that Met-tRNAi (Met) inspects mRNA from the first nucleotide and that initiation does not have a "blind spot." In assembled initiation complexes, the cap was no longer associated with eIF4E. Omission of eIF4A or disruption of eIF4E-eIF4G-eIF3 interactions converted eIF4E into a specific inhibitor of initiation on capped mRNAs. Taken together, these results are consistent with the model in which eIF4E-eIF4G-eIF3-40S interactions place eIF4E at the leading edge of the 40S subunit, and mRNA is threaded into the mRNA-binding channel such that Met-tRNAi (Met) can inspect it from the first nucleotide. Before entering, eIF4E likely dissociates from the cap to overcome steric hindrance. We also found that the m(7)G cap specifically interacts with eIF3l.

Inhibition of translation by IFIT family members is determined by their ability to interact selectively with the 5'-terminal regions of cap0-, cap1- and 5'ppp- mRNAs.

Ribosomal recruitment of cellular mRNAs depends on binding of eIF4F to the mRNA's 5'-terminal 'cap'. The minimal 'cap0' consists of N7-methylguanosine linked to the first nucleotide via a 5'-5' triphosphate (ppp) bridge. Cap0 is further modified by 2'-O-methylation of the next two riboses, yielding 'cap1' (m7GpppNmN) and 'cap2' (m7GpppNmNm). However, some viral RNAs lack 2'-O-methylation, whereas others contain only ppp- at their 5'-end. Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed effectors of innate immunity that inhibit viral replication by incompletely understood mechanisms. Here, we investigated the ability of IFIT family members to interact with cap1-, cap0- and 5'ppp- mRNAs and inhibit their translation. IFIT1 and IFIT1B showed very high affinity to cap-proximal regions of cap0-mRNAs (K1/2,app ∼9 to 23 nM). The 2'-O-methylation abrogated IFIT1/mRNA interaction, whereas IFIT1B retained the ability to bind cap1-mRNA, albeit with reduced affinity (K1/2,app ∼450 nM). The 5'-terminal regions of 5'ppp-mRNAs were recognized by IFIT5 (K1/2,app ∼400 nM). The activity of individual IFITs in inhibiting initiation on a specific mRNA was determined by their ability to interact with its 5'-terminal region: IFIT1 and IFIT1B efficiently outcompeted eIF4F and abrogated initiation on cap0-mRNAs, whereas inhibition on cap1- and 5'ppp- mRNAs by IFIT1B and IFIT5 was weaker and required higher protein concentrations.

Zfp281 and Zfp148 control CD4+ T cell thymic development and TH2 functions.

How CD4+ lineage gene expression is initiated in differentiating thymocytes remains poorly understood. Here, we show that the paralog transcription factors Zfp281 and Zfp148 control both this process and cytokine expression by T helper cell type 2 (TH2) effector cells. Genetic, single-cell, and spatial transcriptomic analyses showed that these factors promote the intrathymic CD4+ T cell differentiation of class II major histocompatibility complex (MHC II)-restricted thymocytes, including expression of the CD4+ lineage-committing factor Thpok. In peripheral T cells, Zfp281 and Zfp148 promoted chromatin opening at and expression of TH2 cytokine genes but not of the TH2 lineage-determining transcription factor Gata3. We found that Zfp281 interacts with Gata3 and is recruited to Gata3 genomic binding sites at loci encoding Thpok and TH2 cytokines. Thus, Zfp148 and Zfp281 collaborate with Gata3 to promote CD4+ T cell development and TH2 cell responses.

Urine Single-Cell RNA Sequencing in Focal Segmental Glomerulosclerosis Reveals Inflammatory Signatures.

INTRODUCTION: Individuals with focal segmental glomerular sclerosis (FSGS) typically undergo kidney biopsy only once, which limits the ability to characterize kidney cell gene expression over time. METHODS: We used single-cell RNA sequencing (scRNA-seq) to explore disease-related molecular signatures in urine cells from subjects with FSGS. We collected 17 urine samples from 12 FSGS subjects and captured these as 23 urine cell samples. The inflammatory signatures from renal epithelial and immune cells were evaluated in bulk gene expression data sets of FSGS and minimal change disease (MCD) (The Nephrotic Syndrome Study Network [NEPTUNE] study) and an immune single-cell data set from lupus nephritis (Accelerating Medicines Partnership). RESULTS: We identified immune cells, predominantly monocytes, and renal epithelial cells in the urine. Further analysis revealed 2 monocyte subtypes consistent with M1 and M2 monocytes. Shed podocytes in the urine had high expression of marker genes for epithelial-to-mesenchymal transition (EMT). We selected the 16 most highly expressed genes from urine immune cells and 10 most highly expressed EMT genes from urine podocytes as immune signatures and EMT signatures, respectively. Using kidney biopsy transcriptomic data from NEPTUNE, we found that urine cell immune signature and EMT signature genes were more highly expressed in FSGS biopsies compared with MCD biopsies. CONCLUSION: The identification of monocyte subsets and podocyte expression signatures in the urine samples of subjects with FSGS suggests that urine cell profiling might serve as a diagnostic and prognostic tool in nephrotic syndrome. Furthermore, this approach may aid in the development of novel biomarkers and identifying personalized therapies targeting particular molecular pathways in immune cells and podocytes.

Location and conformation of pantothenate and its derivatives in Mycobacterium tuberculosis pantothenate kinase: insights into enzyme action.

Previous studies of complexes of Mycobacterium tuberculosis PanK (MtPanK) with nucleotide diphosphates and nonhydrolysable analogues of nucleoside triphosphates in the presence or the absence of pantothenate established that the enzyme has dual specificity for ATP and GTP, revealed the unusual movement of ligands during enzyme action and provided information on the effect of pantothenate on the location and conformation of the nucleotides at the beginning and the end of enzyme action. The X-ray analyses of the binary complexes of MtPanK with pantothenate, pantothenol and N-nonylpantothenamide reported here demonstrate that in the absence of nucleotide these ligands occupy, with a somewhat open conformation, a location similar to that occupied by phosphopantothenate in the `end' complexes, which differs distinctly from the location of pantothenate in the closed conformation in the ternary `initiation' complexes. The conformation and the location of the nucleotide were also different in the initiation and end complexes. An invariant arginine appears to play a critical role in the movement of ligands that takes place during enzyme action. The work presented here completes the description of the locations and conformations of nucleoside diphosphates and triphosphates and pantothenate in different binary and ternary complexes, and suggests a structural rationale for the movement of ligands during enzyme action. The present investigation also suggests that N-alkylpantothenamides could be phosphorylated by the enzyme in the same manner as pantothenate.

A multicenter study benchmarking single-cell RNA sequencing technologies using reference samples.

Comparing diverse single-cell RNA sequencing (scRNA-seq) datasets generated by different technologies and in different laboratories remains a major challenge. Here we address the need for guidance in choosing algorithms leading to accurate biological interpretations of varied data types acquired with different platforms. Using two well-characterized cellular reference samples (breast cancer cells and B cells), captured either separately or in mixtures, we compared different scRNA-seq platforms and several preprocessing, normalization and batch-effect correction methods at multiple centers. Although preprocessing and normalization contributed to variability in gene detection and cell classification, batch-effect correction was by far the most important factor in correctly classifying the cells. Moreover, scRNA-seq dataset characteristics (for example, sample and cellular heterogeneity and platform used) were critical in determining the optimal bioinformatic method. However, reproducibility across centers and platforms was high when appropriate bioinformatic methods were applied. Our findings offer practical guidance for optimizing platform and software selection when designing an scRNA-seq study.

T Cell-Mediated Antitumor Immunity Cooperatively Induced By TGFßR1 Antagonism and Gemcitabine Counteracts Reformation of the Stromal Barrier in Pancreatic Cancer.

The desmoplastic stroma of pancreatic cancers forms a physical barrier that impedes intratumoral drug delivery. Attempts to modulate the desmoplastic stroma to increase delivery of administered chemotherapy have not shown positive clinical results thus far, and preclinical reports in which chemotherapeutic drugs were coadministered with antistromal therapies did not universally demonstrate increased genotoxicity despite increased intratumoral drug levels. In this study, we tested whether TGFß antagonism can break the stromal barrier, enhance perfusion and tumoral drug delivery, and interrogated cellular and molecular mechanisms by which the tumor prevents synergism with coadministered gemcitabine. TGFß inhibition in genetically engineered murine models (GEMM) of pancreas cancer enhanced tumoral perfusion and increased intratumoral gemcitabine levels. However, tumors rapidly adapted to TGFß-dependent stromal modulation, and intratumoral perfusion returned to pre-treatment levels upon extended TGFß inhibition. Perfusion was governed by the phenotypic identity and distribution of cancer-associated fibroblasts (CAF) with the myelofibroblastic phenotype (myCAFs), and myCAFs which harbored unique genomic signatures rapidly escaped the restricting effects of TGFß inhibition. Despite the reformation of the stromal barrier and reversal of initially increased intratumoral exposure levels, TGFß inhibition in cooperation with gemcitabine effectively suppressed tumor growth via cooperative reprogramming of T regulatory cells and stimulation of CD8 T cell-mediated antitumor activity. The antitumor activity was further improved by the addition of anti-PD-L1 immune checkpoint blockade to offset adaptive PD-L1 upregulation induced by TGFß inhibition. These findings support the development of combined antistroma anticancer therapies capable of impacting the tumor beyond the disruption of the desmoplastic stroma as a physical barrier to improve drug delivery.

Mannose receptor (CD206) activation in tumor-associated macrophages enhances adaptive and innate antitumor immune responses.

Solid tumors elicit a detectable immune response including the infiltration of tumor-associated macrophages (TAMs). Unfortunately, this immune response is co-opted into contributing toward tumor growth instead of preventing its progression. We seek to reestablish an antitumor immune response by selectively targeting surface receptors and endogenous signaling processes of the macrophage subtypes driving cancer progression. RP-182 is a synthetic 10-mer amphipathic analog of host defense peptides that selectively induces a conformational switch of the mannose receptor CD206 expressed on TAMs displaying an M2-like phenotype. RP-182-mediated activation of this receptor in human and murine M2-like macrophages elicits a program of endocytosis, phagosome-lysosome formation, and autophagy and reprograms M2-like TAMs to an antitumor M1-like phenotype. In syngeneic and autochthonous murine cancer models, RP-182 suppressed tumor growth, extended survival, and was an effective combination partner with chemo- or immune checkpoint therapy. Antitumor activity of RP-182 was also observed in CD206high patient-derived xenotransplantation models. Mechanistically, via selective reduction of immunosuppressive M2-like TAMs, RP-182 improved adaptive and innate antitumor immune responses, including increased cancer cell phagocytosis by reprogrammed TAMs.

Mycobacterium tuberculosis pantothenate kinase: possible changes in location of ligands during enzyme action.

The crystal structures of complexes of Mycobacterium tuberculosis pantothenate kinase with the following ligands have been determined: (i) citrate; (ii) the nonhydrolysable ATP analogue AMPPCP and pantothenate (the initiation complex); (iii) ADP and phosphopantothenate resulting from phosphorylation of pantothenate by ATP in the crystal (the end complex); (iv) ATP and ADP, each with half occupancy, resulting from a quick soak of crystals in ATP (the intermediate complex); (v) CoA; (vi) ADP prepared by soaking and cocrystallization, which turned out to have identical structures, and (vii) ADP and pantothenate. Solution studies on CoA binding and catalytic activity have also been carried out. Unlike in the case of the homologous Escherichia coli enzyme, AMPPCP and ADP occupy different, though overlapping, locations in the respective complexes; the same is true of pantothenate in the initiation complex and phosphopantothenate in the end complex. The binding site of MtPanK is substantially preformed, while that of EcPanK exhibits considerable plasticity. The difference in the behaviour of the E. coli and M. tuberculosis enzymes could be explained in terms of changes in local structure resulting from substitutions. It is unusual for two homologous enzymes to exhibit such striking differences in action. Therefore, the results have to be treated with caution. However, the changes in the locations of ligands exhibited by M. tuberculosis pantothenate kinase are remarkable and novel.

Genome Assembly and Annotation of the Trichoplusia ni Tni-FNL Insect Cell Line Enabled by Long-Read Technologies.

BACKGROUND: Trichoplusiani derived cell lines are commonly used to enable recombinant protein expression via baculovirus infection to generate materials approved for clinical use and in clinical trials. In order to develop systems biology and genome engineering tools to improve protein expression in this host, we performed de novo genome assembly of the Trichoplusiani-derived cell line Tni-FNL. METHODS: By integration of PacBio single-molecule sequencing, Bionano optical mapping, and 10X Genomics linked-reads data, we have produced a draft genome assembly of Tni-FNL. RESULTS: Our assembly contains 280 scaffolds, with a N50 scaffold size of 2.3 Mb and a total length of 359 Mb. Annotation of the Tni-FNL genome resulted in 14,101 predicted genes and 93.2% of the predicted proteome contained recognizable protein domains. Ortholog searches within the superorder Holometabola provided further evidence of high accuracy and completeness of the Tni-FNL genome assembly. CONCLUSIONS: This first draft Tni-FNL genome assembly was enabled by complementary long-read technologies and represents a high-quality, well-annotated genome that provides novel insight into the complexity of this insect cell line and can serve as a reference for future large-scale genome engineering work in this and other similar recombinant protein production hosts.

An accurately preorganized IRES RNA structure enables eIF4G capture for initiation of viral translation.

Many viruses bypass canonical cap-dependent translation in host cells by using internal ribosomal entry sites (IRESs) in their transcripts; IRESs hijack initiation factors for the assembly of initiation complexes. However, it is currently unknown how IRES RNAs recognize initiation factors that have no endogenous RNA binding partners; in a prominent example, the IRES of encephalomyocarditis virus (EMCV) interacts with the HEAT-1 domain of eukaryotic initiation factor 4G (eIF4G). Here we report the solution structure of the J-K region of this IRES and show that its stems are precisely organized to position protein-recognition bulges. This multisite interaction mechanism operates on an all-or-nothing principle in which all domains are required. This preorganization is accomplished by an 'adjuster module': a pentaloop motif that acts as a dual-sided docking station for base-pair receptors. Because subtle changes in the orientation abrogate protein capture, our study highlights how a viral RNA acquires affinity for a target protein.

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of pantothenate kinase from Mycobacterium tuberculosis.

Pantothenate kinase is an essential enzyme in the bacterial life cycle. It catalyzes the phosphorylation of pantothenate (vitamin B5) to 4'-phosphopantothenate, the first step in the coenzyme A biosynthetic pathway. The enzyme from Mycobacterium tuberculosis, MW 35.7 kDa, has been cloned, expressed, purified and crystallized in two different trigonal crystal forms, both belonging to space group P3(1)21. Two complete data sets of resolution 2.5 A (form I) and 2.9 A (form II) from crystals with unit-cell parameters a = b = 78.3, c = 115.45 A and a = b = 107.63, c = 89.85 A, respectively, were collected at room temperature on a home X-ray source. Structures of both crystal forms were solved for one subunit in the asymmetric unit by molecular replacement.

Widespread distribution and structural diversity of Type IV IRESs in members of Picornaviridae.

Picornavirus genomes contain internal ribosomal entry sites (IRESs) that promote end-independent translation initiation. Five structural classes of picornavirus IRES have been identified, but numerous IRESs remain unclassified. Here, previously unrecognized Type IV IRESs were identified in members of three proposed picornavirus genera (Limnipivirus, Pasivirus, Rafivirus) and four recognized genera (Kobuvirus, Megrivirus, Sapelovirus, Parechovirus). These IRESs are ~230-420 nucleotides long, reflecting heterogeneity outside a common structural core. Closer analysis yielded insights into evolutionary processes that have shaped contemporary IRESs. The presence of related IRESs in diverse genera supports the hypothesis that they are heritable genetic elements that spread by horizontal gene transfer. Recombination likely also accounts for the exchange of some peripheral subdomains, suggesting that IRES evolution involves incremental addition of elements to a pre-existing core. Nucleotide conservation is concentrated in ribosome-binding sites, and at the junction of helical domains, likely to ensure orientation of subdomains in an active conformation.

Calcium/calmodulin dependent protein kinase II bound to NMDA receptor 2B subunit exhibits increased ATP affinity and attenuated dephosphorylation.

Calcium/calmodulin dependent protein kinase II (CaMKII) is implicated to play a key role in learning and memory. NR2B subunit of N-methyl-D-aspartate receptor (NMDAR) is a high affinity binding partner of CaMKII at the postsynaptic membrane. NR2B binds to the T-site of CaMKII and modulates its catalysis. By direct measurement using isothermal titration calorimetry (ITC), we show that NR2B binding causes about 11 fold increase in the affinity of CaMKII for ATPγS, an analogue of ATP. ITC data is also consistent with an ordered binding mechanism for CaMKII with ATP binding the catalytic site first followed by peptide substrate. We also show that dephosphorylation of phospho-Thr(286)-α-CaMKII is attenuated when NR2B is bound to CaMKII. This favors the persistence of Thr(286) autophosphorylated state of CaMKII in a CaMKII/phosphatase conjugate system in vitro. Overall our data indicate that the NR2B- bound state of CaMKII attains unique biochemical properties which could help in the efficient functioning of the proposed molecular switch supporting synaptic memory.

M. tuberculosis pantothenate kinase: dual substrate specificity and unusual changes in ligand locations.

Kinetic measurements of enzyme activity indicate that type I pantothenate kinase from Mycobacterium tuberculosis has dual substrate specificity for ATP and GTP, unlike the enzyme from Escherichia coli, which shows a higher specificity for ATP. A molecular explanation for the difference in the specificities of the two homologous enzymes is provided by the crystal structures of the complexes of the M. tuberculosis enzyme with (1) GMPPCP and pantothenate, (2) GDP and phosphopantothenate, (3) GDP, (4) GDP and pantothenate, (5) AMPPCP, and (6) GMPPCP, reported here, and the structures of the complexes of the two enzymes involving coenzyme A and different adenyl nucleotides reported earlier. The explanation is substantially based on two critical substitutions in the amino acid sequence and the local conformational change resulting from them. The structures also provide a rationale for the movement of ligands during the action of the mycobacterial enzyme. Dual specificity of the type exhibited by this enzyme is rare. The change in locations of ligands during action, observed in the case of the M. tuberculosis enzyme, is unusual, so is the striking difference between two homologous enzymes in the geometry of the binding site, locations of ligands, and specificity. Furthermore, the dual specificity of the mycobacterial enzyme appears to have been caused by a biological necessity.

The role of UPF0157 in the folding of M. tuberculosis dephosphocoenzyme A kinase and the regulation of the latter by CTP.

BACKGROUND: Targeting the biosynthetic pathway of Coenzyme A (CoA) for drug development will compromise multiple cellular functions of the tubercular pathogen simultaneously. Structural divergence in the organization of the penultimate and final enzymes of CoA biosynthesis in the host and pathogen and the differences in their regulation mark out the final enzyme, dephosphocoenzyme A kinase (CoaE) as a potential drug target. METHODOLOGY/PRINCIPAL FINDINGS: We report here a complete biochemical and biophysical characterization of the M. tuberculosis CoaE, an enzyme essential for the pathogen's survival, elucidating for the first time the interactions of a dephosphocoenzyme A kinase with its substrates, dephosphocoenzyme A and ATP; its product, CoA and an intrinsic yet novel inhibitor, CTP, which helps modulate the enzyme's kinetic capabilities providing interesting insights into the regulation of CoaE activity. We show that the mycobacterial enzyme is almost 21 times more catalytically proficient than its counterparts in other prokaryotes. ITC measurements illustrate that the enzyme follows an ordered mechanism of substrate addition with DCoA as the leading substrate and ATP following in tow. Kinetic and ITC experiments demonstrate that though CTP binds strongly to the enzyme, it is unable to participate in DCoA phosphorylation. We report that CTP actually inhibits the enzyme by decreasing its Vmax. Not surprisingly, a structural homology search for the modeled mycobacterial CoaE picks up cytidylmonophosphate kinases, deoxycytidine kinases, and cytidylate kinases as close homologs. Docking of DCoA and CTP to CoaE shows that both ligands bind at the same site, their interactions being stabilized by 26 and 28 hydrogen bonds respectively. We have also assigned a role for the universal Unknown Protein Family 0157 (UPF0157) domain in the mycobacterial CoaE in the proper folding of the full length enzyme. CONCLUSIONS/SIGNIFICANCE: In view of the evidence presented, it is imperative to assign a greater role to the last enzyme of Coenzyme A biosynthesis in metabolite flow regulation through this critical biosynthetic pathway.

How pantothenol intervenes in Coenzyme-A biosynthesis of Mycobacterium tuberculosis.

Coenzyme A is an indispensable cofactor for all organisms and holds a central position in a number of pathways. Prokaryotic enzymes involved in the synthesis of CoA are quite different from their mammalian counterparts; hence, they are good targets for the development of antimicrobials to treat many diseases. There are antimicrobials that act by inhibiting CoA biosynthesis. It has been suggested that pantothenol exhibits antibacterial activity by competitively inhibiting pantothenate kinase, a key regulatory enzyme for CoA synthesis. Contrary to these suggestions, in this paper, we demonstrate that pantothenol acts as a substrate for Mycobacterium tuberculosis and Escherichia coli pantothenate kinases. The product, 4'-phosphopantothenol, thus formed inhibits competitively the utilization of 4'-phosphopantothenate by CoaBC. Thus, it is the failure of CoaBC to utilize 4'-phosphopantothenol as a substrate that accounts for the bactericidal activity of pantothenol.

A double helix is the repeating unit in a luminescent calcium 5-aminoisophthalate supramolecular edifice with water-filled hexagonal channels.

Calcium 5-aminoisophthalate {[Ca(AIP)(OH2)4](H2O)} proportional, variant (1), synthesized from Ca(OH)2 and H2AIP, exists as a double helix. Hydrogen-bonding interactions between metal-bound water molecules, carboxylate oxygen, and the -NH2 group result in the formation of a supramolecular assembly, where the double helices are connected to each other to form hexagonal channels filled with water molecules.

Invariance and variability in bacterial PanK: a study based on the crystal structure of Mycobacterium tuberculosis PanK.

Pantothenate kinase (PanK) is a ubiquitous and essential enzyme that catalyzes the first step of the universal coenzyme A biosynthetic pathway. In this step, pantothenate (vitamin B(5)) is converted to 4'-phosphopantothenate, which subsequently forms coenzyme A in four enzymatic steps. The complex of this enzyme from Mycobacterium tuberculosis (MtPanK) with a derivative of the feedback inhibitor coenzyme A has been crystallized in two forms and its structure solved. The structure was refined in both forms using room-temperature and low-temperature X-ray data. In both forms, the MtPanK subunit has a mononucleotide-binding fold with a seven-stranded central beta-sheet and helices on either side. However, there is a small though significant difference in subunit association between the two forms. The structure is also grossly similar to the enzyme from Escherichia coli. The active-site pocket and the dimeric interface are on two opposite sides of the PanK subunit. The enzymes from M. tuberculosis and E. coli exhibit several differences, particularly at the dimeric interface. On the other hand, the coenzyme A-binding region is almost entirely conserved. A delineation of the invariant and variable features of the PanK structure further indicates that the dimeric interface is very variable, while the coenzyme A-binding site is substantially invariant. A sequence alignment involving various bacterial PanKs is in agreement with this conclusion. The strong correlation between structural plasticity, evolutionary conservation and variability and function exhibited by the molecule could be important in the design of species-specific inhibitors of the enzyme.

Broad substrate stereospecificity of the Mycobacterium tuberculosis 7-keto-8-aminopelargonic acid synthase: Spectroscopic and kinetic studies.

Biotin is an essential enzyme cofactor required for carboxylation and transcarboxylation reactions. The absence of the biotin biosynthesis pathway in humans suggests that it can be an attractive target for the development of novel drugs against a number of pathogens. 7-Keto-8-aminopelargonic acid (KAPA) synthase (EC 2.3.1.47), the enzyme catalyzing the first committed step in the biotin biosynthesis pathway, is believed to exhibit high substrate stereospecificity. A comparative kinetic characterization of the interaction of the mycobacterium tuberculosis KAPA synthase with both L- AND D-alanine was carried out to investigate the basis of the substrate stereospecificity exhibited by the enzyme. The formation of the external aldimine with D-alanine (k = 82.63 m(-1) s(-1)) is approximately 5 times slower than that with L-alanine (k = 399.4 m(-1) s(-1)). In addition to formation of the external aldimine, formation of substrate quinonoid was also observed upon addition of pimeloyl-CoA to the preformed d-alanine external aldimine complex. However, the formation of this intermediate was extremely slow compared with the substrate quinonoid with L-alanine and pimeloyl-CoA (k = 16.9 x 10(4) m(-1) s(-1)). Contrary to earlier reports, these results clearly show that D-alanine is not a competitive inhibitor but a substrate for the enzyme and thereby demonstrate the broad substrate stereospecificity of the M. tuberculosis KAPA synthase. Further, d-KAPA, the product of the reaction utilizing D-alanine inhibits both KAPA synthase (Ki = 114.83 microm) as well as 7,8-diaminopelargonic acid synthase (IC50 = 43.9 microm), the next enzyme of the pathway.