SUMMARY The mycobacterial biotin protein ligase (MtBPL) globally regulates lipid metabolism in Mtb through the posttranslational biotinylation of acyl coenzyme A carboxylases involved in lipid biosynthesis that catalyze the first step in fatty acid biosynthesis and pyruvate coenzyme A carboxylase, a gluconeogenic enzyme vital for lipid catabolism. Here we describe the design, development and evaluation of a rationally designed bisubstrate inhibitor of MtBPL. This inhibitor displays potent sub-nanomolar enzyme inhibition and antitubercular activity against multi- and extensively drug resistant Mtb strains. We show that the inhibitor decreases in vivo protein biotinylation of key enzymes involved in fatty acid biosynthesis and that the anti-bacterial activity is MtBPL-dependent. Additionally, the gene encoding BPL was found to be essential in M. smegmatis. Finally, the X-ray co-crystal structure of inhibitor bound MtBPL was solved providing detailed insight for further structure-activity analysis. Collectively, these data suggest that MtBPL is a promising target for further antitubercular therapeutic development.

Immobilized and site-specifically labeled proteins are becoming invaluable tools in proteomics. Here, we describe a strategy to attach a desired protein to a solid surface in a covalent, site-specific manner. This approach employs an enzymatic posttranslational modification method to site-specifically label a target protein with an azide; an alternative substrate for protein farnesyl transferase containing an azide group was developed for this purpose. A bio-orthogonal Cu(I)-catalyzed cycloaddition reaction is then used to covalently attach the protein to agarose beads bearing an alkyne functional group. We demonstrate that both the azide incorporation and the capture steps can be performed on either a purified protein target or on a protein present within a complex mixture. This approach involves the use of a four-residue tag which is significantly smaller than most other tags reported to date and results in covalent immobilization of the target protein. Hence it should have significant applicability in protein science.

The challenging task of identifying and studying protein function has been greatly aided by labeling proteins with reporter groups. Here, we present a strategy that utilizes an enzyme that labels a four-residue sequence appended onto the C terminus of a protein, with an alkyne-containing substrate. By using a bio-orthogonal cycloaddition reaction, a fluorophore that carried an azide moiety was then covalently coupled to the alkyne appended on the protein. FRET was used to calculate a Förster (R) distance of 40 A between the eGFP chromophore and the newly appended Texas Red fluorophore. This experimental value is in good agreement with the predicted R value determined by using molecular modeling. The small recognition tag, the high specificity of the enzyme, and the orthogonal nature of the derivatization reaction will make this approach highly useful in protein chemistry.

The human pathogen Acinetobacter baumannii produces a siderophore called acinetobactin that is derived from one molecule each of threonine, histidine, and 2,3-dihydroxybenzoic acid (DHB). The activity of several non-ribosomal peptide synthetase (NRPS) enzymes is used to combine the building blocks into the final molecule. The acinetobactin synthesis pathway initiates with a selfstanding adenylation enzyme, BasE, that activates the DHB molecule and covalently transfers it to the pantetheine cofactor of an aryl-carrier protein of BasF, a strategy that is shared with many siderophore-producing NRPS clusters. In this reaction, DHB reacts with ATP to form the aryl adenylate and pyrophosphate. In a second partial reaction, the DHB is transferred to the carrier protein. Inhibitors of BasE and related enzymes have been identified that prevent growth of bacteria on iron-limiting media. Recently, a new inhibitor of BasE has been identified via highthroughput screening using a fluorescence polarization displacement assay. We present here biochemical and structural studies to examine the binding mode of this inhibitor. The kinetics of the wild-type BasE enzyme is shown and inhibition studies demonstrate that the new compound exhibits competitive inhibition against both ATP and 2,3-dihydroxybenzoate. Structural examination of BasE bound to this inhibitor illustrates a novel binding mode in which the phenyl moiety partially fills the enzyme pantetheine binding tunnel. Structures of rationally designed bisubstrate inhibitors are also presented. KeywordsSiderophore synthesis; X-ray crystallography; Non-Ribosomal Peptide Synthetase; ANL Superfamily; Adenylate-forming enzymes; Acinetobacter baumannii † This work is supported in part by NIH Grant GM-068440 (to A.M.G.) and AI-070219 (to C.C.A). Diffraction data were collected at the Cornell High Energy Synchrotron Source which is supported by the National Science Foundation under award DMR 0225180 and the National Institutes of Health through its National Center for Research Resources under award 5 P41 RR001646-23, and at the Stanford Synchrotron Radiation Lightsource, a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences. * Correspondence to Andrew M. Gulick: Hauptman-Woodward Institute, Department of Structural Biology. University at Buffalo, 700 Ellicott St, Buffalo, NY 14203-1102. Phone (716) . gulick@hwi.buffalo.edu. The structure factors and coordinates of the BasE protein bound to 5 (3O82), 7 (3O83), and 8 (3O84) have been deposited with the Protein Data Bank. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 November 2. NIH-PA Author ManuscriptN...

Adenylation or adenylate-forming enzymes (AEs) are widely found in nature and are responsible for the activation of carboxylic acids to intermediate acyladenylates, which are mixed anhydrides of AMP. In a second reaction, AEs catalyze the transfer of the acyl group of the acyladenylate onto a nucleophilic amino, alcohol, or thiol group of an acceptor molecule leading to amide, ester, and thioester products, respectively. Mycobacterium tuberculosis encodes for more than 60 adenylating enzymes, many of which represent potential drug targets due to their confirmed essentiality or requirement for virulence. Several strategies have been used to develop potent and selective AE inhibitors including high-throughput screening, fragment-based screening, and the rationale design of bisubstrate inhibitors that mimic the acyladenylate. In this review, a comprehensive analysis of the mycobacterial adenylating enzymes will be presented with a focus on the identification of small molecule inhibitors. Specifically, this review will cover the aminoacyl tRNA-synthetases (aaRSs), MenE required for menaquinone synthesis, the FadD family of enzymes including the fatty acyl-AMP ligases (FAAL) and the fatty acyl-CoA ligases (FACLs) involved in lipid metabolism, and the nonribosomal peptide synthetase adenylation enzyme MbtA that is necessary for mycobactin synthesis. Additionally, the enzymes NadE, GuaA, PanC, and MshC involved in the respective synthesis of NAD, guanine, pantothenate, and mycothiol will be discussed as well as BirA that is responsible for biotinylation of the acyl CoA-carboxylases.

DNA has numerous attractive features as a scaffold for nanostructure assembly. Its rigidity, predictable structure, and assembly through complementary hybridization allow DNA to form nanoscale architectures such as cubes, [1] tetrahedra, [2] octahedra, [3,4] and 2D arrays. [5][6][7][8] By introducing proteins into DNA nanostructures, the recognition elements and functionalities that are inherent in proteins can be organized into nanostructured motifs. DNA-scaffolded protein assemblies have been used in immuno-PCR detection methods (PCR = polymerase chain reaction) [9][10][11] to arrange biocatalysts in a series for sequential reactions [12,13] and to organize other nanomaterials. [14] There are currently several methodologies used to link proteins to DNA. Proteins have been assembled onto DNA scaffolds through intervening adapter molecules such as streptavidin [12,[15][16][17][18][19][20] or aptamers. [21,22] Alternately, direct covalent conjugation can be achieved by modification of cysteine or lysine residues [23][24][25][26] or intein modification. [11,27,28] Niemyer and co-workers have employed these protein-DNA conjugates to form fluorescence resonant energy transfer (FRET) systems for use in nanobiotechnology. [29,30] Herein, we demonstrate a fusion-based strategy to regioselectively and covalently label proteins at the C terminus with single-stranded DNA. These protein-oligonucleotide chimeras were then spontaneously assembled into nanoarchitectures by complementary hybridization of the DNA. The covalent attachment strategy described herein yields a short and compact linkage between the protein and DNA molecule that allows for precise control over protein spacing and orientation in the final nanostructure.To achieve selective protein labeling, we use the enzyme protein farnesyltransferase (PFTase) to label a substrate protein containing a C-terminal tetrapeptide tag with an azide-modified isoprenoid diphosphate (1, Scheme 1).

MbtA is an adenylating enzyme from Mycobacterium tuberculosis that catalyzes the first step in the biosynthesis of the mycobactins. A potent bisubstrate inhibitor (Sal-AMS) of MbtA was previously described that displays potent antitubercular activity under iron-replete as well as iron-deficient growth conditions. This finding is surprising since mycobactin biosynthesis is not required under iron-replete conditions and suggests off-target inhibition of additional biochemical pathways. As a first step towards a complete understanding of the mechanism of action of Sal-AMS, we have designed and validated an activity-based probe (ABP) for studying Sal-AMS inhibition in M. tuberculosis. This probe labels pure MbtA as well as MbtA in mycobacterial lysate and labeling can be completely inhibited by preincubation with Sal-AMS. Furthermore, this probe provides a prototypical core scaffold for the creation of ABPs to profile any of the other 66 adenylating enzymes in Mtb or the multitude of adenylating enzymes in other pathogenic bacteria.

A series of 2-aminothiazoles was synthesized based on a HTS scaffold from a whole-cell screen against Mycobacterium tuberculosis (Mtb). The SAR shows the central thiazole moiety and the 2-pyridyl moiety at C-4 of the thiazole are intolerant to modification. However, the N-2 position of the aminothiazole exhibits high flexibility and we successfully improved the antitubercular activity of the initial hit by more than 128-fold through introduction of substituted benzoyl groups at this position. N-(3-Chlorobenzoyl)-4-(2-pyridinyl)-1,3-thiazol-2-amine (55) emerged as one of the most promising analogues with a MIC of 0.024 μM or 0.008 μg/mL in 7H9 media and therapeutic index of nearly ~300. However, 55 is rapidly metabolized by human liver microsomes (t1/2 = 28 min) with metabolism occurring at the invariant aminothiazole moiety and Mtb develops spontaneous resistance with a high frequency of ~10−5.