Marcy Hernick

The Arabidopsis thaliana Myo-Inositol 1-Phosphate Synthase1 Gene Is Required for Myo-inositol Synthesis and Suppression of Cell Death

This work uses functional genomics to delineate the role of the inositol synthesis genes in regulating growth, development, and cell death and reveals a connection between inositol, phosphatidylinositol, and sphingolipids. l-myo-inositol 1-phosphate synthase (MIPS; EC 5.5.1.4) catalyzes the rate-limiting step in the synthesis of myo-inositol, a critical compound in the cell. Plants contain multiple MIPS genes, which encode highly similar enzymes. We characterized the expression patterns of the three MIPS genes in Arabidopsis thaliana and found that MIPS1 is expressed in most cell types and developmental stages, while MIPS2 and MIPS3 are mainly restricted to vascular or related tissues. MIPS1, but not MIPS2 or MIPS3, is required for seed development, for physiological responses to salt and abscisic acid, and to suppress cell death. Specifically, a loss in MIPS1 resulted in smaller plants with curly leaves and spontaneous production of lesions. The mips1 mutants have lower myo-inositol, ascorbic acid, and phosphatidylinositol levels, while basal levels of inositol (1,4,5)P3 are not altered in mips1 mutants. Furthermore, mips1 mutants exhibited elevated levels of ceramides, sphingolipid precursors associated with cell death, and were complemented by a MIPS1-green fluorescent protein (GFP) fusion construct. MIPS1-, MIPS2-, and MIPS3-GFP each localized to the cytoplasm. Thus, MIPS1 has a significant impact on myo-inositol levels that is critical for maintaining levels of ascorbic acid, phosphatidylinositol, and ceramides that regulate growth, development, and cell death.

UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine Deacetylase Functions through a General Acid-Base Catalyst Pair Mechanism

UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc-dependent enzyme that catalyzes the deacetylation of UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine to form UDP-3-O-(R-hydroxymyristoyl)glucosamine and acetate. The structural similarity of the active site of LpxC to metalloproteases led to the proposal that LpxC functions via a metalloprotease-like mechanism. The pH dependence of kcat/Km catalyzed by Escherichia coli and Aquifex aeolicus LpxC displayed a bell-shaped curve (EcLpxC yields apparent pKa values of 6.4 ± 0.1 and 9.1 ± 0.1), demonstrating that at least two ionizations are important for maximal activity. Metal substitution and mutagenesis experiments suggest that the basic limb of the pH profile is because of deprotonation of a zinc-coordinated group such as the zinc-water molecule, whereas the acidic limb of the pH profile is caused by protonation of either Glu78 or His265. Furthermore, the magnitude of the activity decreases and synergy observed for the active site mutants suggest that Glu78 and His265 act as a general acid-base catalyst pair. Crystal structures of LpxC complexed with cacodylate or palmitate demonstrate that both Glu78 and His265 hydrogen-bond with the same oxygen atom of the tetrahedral intermediate and the product carboxylate. These structural features suggest that LpxC catalyzes deacetylation by using Glu78 and His265 as a general acid-base pair and the zinc-bound water as a nucleophile.

Active site metal ion in UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) switches between Fe(II) and Zn(II) depending on cellular conditions.

UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the deacetylation of UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine to form UDP-3-O-myristoylglucosamine and acetate in Gram-negative bacteria. This second, and committed, step in lipid A biosynthesis is a target for antibiotic development. LpxC was previously identified as a mononuclear Zn(II) metalloenzyme; however, LpxC is 6–8-fold more active with the oxygen-sensitive Fe(II) cofactor (Hernick, M., Gattis, S. G., Penner-Hahn, J. E., and Fierke, C. A. (2010) Biochemistry 49, 2246–2255). To analyze the native metal cofactor bound to LpxC, we developed a pulldown method to rapidly purify tagged LpxC under anaerobic conditions. The metal bound to LpxC purified from Escherichia coli grown in minimal medium is mainly Fe(II). However, the ratio of iron/zinc bound to LpxC varies with the metal content of the medium. Furthermore, the iron/zinc ratio bound to native LpxC, determined by activity assays, has a similar dependence on the growth conditions. LpxC has significantly higher affinity for Zn(II) compared with Fe(II) with KD values of 60 ± 20 pm and 110 ± 40 nm, respectively. However, in vivo concentrations of readily exchangeable iron are significantly higher than zinc, suggesting that Fe(II) is the thermodynamically favored metal cofactor for LpxC under cellular conditions. These data indicate that LpxC expressed in E. coli grown in standard medium predominantly exists as the Fe(II)-enzyme. However, the metal cofactor in LpxC can switch between iron and zinc in response to perturbations in available metal ions. This alteration may be important for regulating the LpxC activity upon changes in environmental conditions and may be a general mechanism of regulating the activity of metalloenzymes.

Activation of Escherichia coli UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine Deacetylase by Fe2+ Yields a More Efficient Enzyme with Altered Ligand Affinity

The metal-dependent deacetylase UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC) catalyzes the first committed step in lipid A biosynthesis, the hydrolysis of UDP-3-O-myristoyl-N-acetylglucosamine to form UDP-3-O-myristoylglucosamine and acetate. Consequently, LpxC is a target for the development of antibiotics, nearly all of which coordinate the active site metal ion. Here we examine the ability of Fe(2+) to serve as a cofactor for wild-type Escherichia coli LpxC and a mutant enzyme (EcC63A), in which one of the ligands for the inhibitory metal binding site has been removed. LpxC exhibits higher activity (6-8-fold) with a single bound Fe(2+) as the cofactor compared to Zn(2+)-LpxC; both metalloenzymes have a bell-shaped dependence on pH with similar pK(a) values, indicating that at least two ionizations are important for maximal activity. X-ray absorption spectroscopy experiments suggest that the catalytic metal ion bound to Fe(2+)-EcLpxC is five-coordinate, suggesting that catalytic activity may correlate with coordination number. Furthermore, the ligand affinity of Fe(2+)-LpxC compared to the Zn(2+) enzyme is altered by up to 6-fold. In contrast to Zn(2+)-LpxC, the activity of Fe(2+)-LpxC is redox-sensitive, and a time-dependent decrease in activity is observed under aerobic conditions. The LpxC activity of crude E. coli cell lysates is also aerobically sensitive, consistent with the presence of Fe(2+)-LpxC. These data indicate that EcLpxC can use either Fe(2+) or Zn(2+) to activate catalysis in vitro and possibly in vivo, which may allow LpxC to function in E. coli grown under different environmental conditions.

The Activity and Cofactor Preferences of N-Acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside Deacetylase (MshB) Change Depending on Environmental Conditions

Actinomycetes, such as Mycobacterium species, are Gram-positive bacteria that utilize the small molecule mycothiol (MSH) as their primary reducing agent. Consequently, the enzymes involved in MSH biosynthesis are targets for drug development. The metal-dependent enzyme N-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside deacetylase (MshB) catalyzes the hydrolysis of N-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside to form 1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside and acetate, the fourth overall step in MSH biosynthesis. Inhibitors of metalloenzymes typically contain a group that binds to the active site metal ion; thus, a comprehensive understanding of the native cofactor(s) of metalloenzymes is critical for the development of biologically effective inhibitors. Herein, we examined the effect of metal ions on the overall activity of MshB and probed the identity of the native cofactor. We found that the activity of MshB follows the trend Fe2+ > Co2+ > Zn2+ > Mn2+ and Ni2+. Additionally, our results show that the identity of the cofactor bound to purified MshB is highly dependent on the purification conditions used (aerobic versus anaerobic), as well as the metal ion content of the medium during protein expression. MshB prefers Fe2+ under anaerobic conditions regardless of the metal ion content of the medium and switches between Fe2+ and Zn2+ under aerobic conditions as the metal content of the medium is altered. These results indicate that the cofactor bound to MshB under biological conditions is dependent on environmental conditions, suggesting that MshB may be a cambialistic metallohydrolase that contains a dynamic cofactor. Consequently, biologically effective inhibitors will likely need to dually target Fe2+-MshB and Zn2+-MshB.

Residue Ionization in LpxC Directly Observed by 67Zn NMR Spectroscopy

The pH dependence of the solid-state (67)Zn NMR lineshapes has been measured for both the wild type (WT) and the H265A mutant of Aquifex aeolicus LpxC, each in the absence of substrate (resting state). The (67)Zn NMR spectrum of WT LpxC at pH 6 (prepared at 0 degrees C) contains two overlapping quadrupole lineshapes with C q values of 10 and 12.9 MHz, while the spectrum measured for the sample prepared at a pH near 9 (at 0 degrees C) is dominated by the appearance of a third species with a C q of 14.3 MHz. These findings are consistent with the two p K a values previously observed by the bell-shaped dependence of the LpxC-catalyzed reaction. On the basis of comparison of the experimental results with predictions from quantum mechanical/molecular mechanical (QM/MM) modeling, we suggest that p K a1 (low pH) represents the ionization of Glu78 and p K a2 (high pH) reflects the ionization of another active site residue located near the zinc ion, such as His265. These results are also consistent with water being bound to the Zn (2+) ion throughout this pH range. The (67)Zn NMR spectra of the H265A mutant appear to be pH independent, with a C q of 9.55 MHz being sufficient to describe both low- and high-pH data. The QM/MM models of the H265A mutant suggest that over this pH range water is bound to the zinc ion while Glu78 is protonated.

A fluorescence-based assay for measuring N-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside deacetylase activity

Here we report a new fluorescence-based assay for measuring MshB (N-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside deacetylase) activity. The current assay for measuring MshB activity requires the fluorescent labeling of reaction mixtures and subsequent analysis using high-performance liquid chromatography (HPLC), resulting in a significant amount of processing time per sample. Here we describe a more rapid fluorescnce-based assay for the measurement of MshB activity that does not require HPLC analysis and can be carried out in multiwell plates. This fluorescamine (FSA)-based assay was used to determine the steady-state parameters for the deacetylation of N-acetyl-glucosamine (GlcNAc) by MshB, and the results from these experiments support the hypothesis that the inositol moiety primarily contributes to the affinity of GlcNAc-Ins (N-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside) for MshB. The rapid nature of this assay will aid efforts toward a more detailed biochemical characterization of MshB. Furthermore, because this assay relies on the formation of a primary amine, it could be adapted to measure the activity of mycothiol-S-conjugate amidase, a metal-dependent amidase that is a potential drug target involved in the mycothiol detoxification pathway.

Mycothiol: a target for potentiation of rifampin and other antibiotics against Mycobacterium tuberculosis

Actinomycetes, including Mycobacterium species, are Gram-positive bacteria that use the small molecule mycothiol (MSH) as their primary reducing agent and in the detoxification of xenobiotics. Due to these important functions, MSH is a potential target for the development of antibiotics for the treatment of tuberculosis. This review summarizes the progress to date on the viability of enzymes involved in MSH biosynthesis and MSH-dependent detoxification as drug targets, biochemical characterization of target enzymes (structure, mechanism and substrate specificity) and development of MSH biosynthesis and MSH-dependent detoxification enzyme inhibitors. In addition, the ability of MSH to influence the sensitivity of mycobacteria to existing antibiotics and potential of MSH biosynthesis and MSH-dependent detoxification enzyme inhibitors to modulate the activity of existing antibiotics are described.

Examination of Mechanism of N-Acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside Deacetylase (MshB) Reveals Unexpected Role for Dynamic Tyrosine

Background: MshB is a metal-dependent deacetylase involved in mycothiol biosynthesis. Results: The reaction proceeds via a general acid-base pair mechanism and uses a dynamic Tyr that modulates substrate binding, chemistry, and product release. Conclusion: The catalytic mechanism differs from a prototypical metalloprotease mechanism. Significance: Key side chains identified in these studies can be targeted for inhibitor development. Actinomycetes are a group of Gram-positive bacteria that includes pathogenic mycobacterial species, such as Mycobacterium tuberculosis. These organisms do not have glutathione and instead utilize the small molecule mycothiol (MSH) as their primary reducing agent and for the detoxification of xenobiotics. Due to these important functions, enzymes involved in MSH biosynthesis and MSH-dependent detoxification are targets for drug development. The metal-dependent deacetylase N-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside deacetylase (MshB) catalyzes the hydrolysis of N-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside to form 1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside and acetate in MSH biosynthesis. Herein we examine the chemical mechanism of MshB. We demonstrate that the side chains of Asp-15, Tyr-142, His-144, and Asp-146 are important for catalytic activity. We show that NaF is an uncompetitive inhibitor of MshB, consistent with a metal-water/hydroxide functioning as the reactive nucleophile in the catalytic mechanism. We have previously shown that MshB activity has a bell-shaped dependence on pH with pKa values of ∼7.3 and 10.5 (Huang, X., Kocabas, E. and Hernick, M. (2011) J. Biol. Chem. 286, 20275–20282). Mutagenesis experiments indicate that the observed pKa values reflect ionization of Asp-15 and Tyr-142, respectively. Together, findings from our studies suggest that MshB functions through a general acid-base pair mechanism with the side chain of Asp-15 functioning as the general base catalyst and His-144 serving as the general acid catalyst, whereas the side chain of Tyr-142 probably assists in polarizing substrate/stabilizing the oxyanion intermediate. Additionally, our results indicate that Tyr-142 is a dynamic side chain that plays key roles in catalysis, modulating substrate binding, chemistry, and product release.

Automated docking studies provide insights into molecular determinants of ligand recognition by N-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside deacetylase (MshB)

The metal-dependent deacetylase N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside deacetylase (MshB) catalyzes the deacetylation of N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcNAc-Ins), the committed step in mycothiol (MSH) biosynthesis. MSH is the thiol redox buffer used by mycobacteria to protect against oxidative damage and is involved in the detoxification of xenobiotics. As such, MshB is a target for the discovery of new drugs to treat tuberculosis (TB). While MshB substrate specificity and inhibitor activity have been probed extensively using enzyme kinetics, information regarding the molecular basis for the observed differences in substrate specificity and inhibitor activity is lacking. Herein we begin to examine the molecular determinants of MshB substrate specificity using automated docking studies with a set of known MshB substrates. Results from these studies offer insights into molecular recognition by MshB via identification of side chains and dynamic loops that may play roles in ligand binding. Additionally, results from these studies suggest that a hydrophobic cavity adjacent to the active site may be one important determinant of MshB substrate specificity. Importantly, this hydrophobic cavity may be advantageous for the design of MshB inhibitors with high affinity and specificity as potential TB drugs.