Functional Characterization of Human Carbonic Anhydrase II Variants with Altered Zinc Binding Sites

2 as their primary reducing agent and in xenobiotic metabolism for the detoxification of drugs and other toxins (1-4). MSH is likely to be critical for the survival of mycobacteria inside activated macrophages, where the mycobacteria are subjected to oxidative bursts. Consequently, the enzymes involved in MSH biosynthesis and detoxification (Fig. 1A), including the metalloenzymes N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-␣-D-glucopyranoside deacetylase (MshB) and MSH-conjugate amidase, are targets for the development of antibiotics for the treatment of diseases such as tuberculosis (5-10).The enzyme MshB catalyzes the hydrolysis of (GlcN-Ins) and acetate, the fourth overall step in MSH biosynthesis (rate-limiting step) (11). MshB is an attractive drug target because it is a metalloenzyme; there are past successes in targeting metalloenzymes, including inhibitors of carbonic anhydrase, matrix metalloproteases, and angiotensinconverting enzyme (12-15). Inhibitors of metalloenzymes typically contain a group that binds to the catalytic metal ion. Consequently, a comprehensive understanding of metalloenzyme cofactor preferences is necessary for the development of potent and specific metalloenzyme inhibitors.MshB was previously identified as a Zn 2ϩ -dependent enzyme based on the observations that the enzyme copurifies with Zn 2ϩ (Fig. 1B) and that the enzyme activity is reversibly inhibited by treatment with 1,10-phenanthroline (16 -18). On the basis of the structure of the enzyme active site, MshB is thought to catalyze the hydrolysis of GlcNAc-Ins via one of two potential chemical mechanisms using general acid-base catalysis (GABC) (19). One possible mechanism uses a single bifunctional GABC to facilitate the hydrolysis of GlcNAc-Ins, whereas the other uses a GABC pair to carry out this reaction. However, Fe 2ϩ was not examined as a potential cofactor in these experiments. Furthermore, MshB was purified using zinc immobilized metal ion affinity chromatography (IMAC) under aerobic conditions, which is biased toward zinc incorporation into metalloenzymes (16). Purified MshB contains nickel (0.82 eq) when purified using nickel IMAC (aerobic conditions) (16). There have been several examples over the last decade of Fe 2ϩ -enzymes being misidentified as exclusive Zn 2ϩ -enzymes, including peptide deformylase, S-ribosylhomocysteinase (LuxS), UDP-3-O-(R-3-hydroxymyristoyl)-Nacetylglucosamine deacetylase (LpxC), and possibly histone deacetylase-8 (HDAC8) (20 -27). In all these enzymes, the Fe 2ϩ cofactor is either exclusively preferred or is preferred over Zn 2ϩ under certain environmental conditions. The

Section: Cofactor Preferences Are Determined By Metal Ionmentioning

confidence: 99%

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

Huang

Kocabas

Hernick

2011

“…Furthermore, it is the first time a metal ion site has been created in a region outside of the seven transmembrane regions of a GPCR and the loops connecting these. As it was to be expected from the existence of numerous endogenous metal ion binding sites in proteins (27)(28)(29)48), the use of zinc as a structural probe in GPCRs can clearly be extended to regions outside of the "confinement" of the membrane. While not all ligands for Zn 2ϩ in K260H mGluR1 may have been identified, the findings in this study offer valuable insight into the mechanism of ATD closure and family C receptor activation, identify regions in the ATD as interesting targets from a medicinal chemistry point of view, and strongly support our previously published model of the ATD of mGluR1 (11).…”

Section: H]-quis Binding To Wt and Mutant Mglurlb Receptorsmentioning

confidence: 99%

Construction of a High Affinity Zinc Binding Site in the Metabotropic Glutamate Receptor mGluR1

Jensen

Sheppard

Jensen

et al. 2001

The metabotropic glutamate receptors (mGluRs) belong to family C of the G-protein-coupled receptor (GPCR) superfamily. The receptors are characterized by having unusually long amino-terminal domains (ATDs), to which agonist binding has been shown to take place. Previously, we have constructed a molecular model of the ATD of mGluR1 based on a weak amino acid sequence similarity with a bacterial periplasmic binding protein. The ATD consists of two globular lobes, which are speculated to contract from an "open" to a "closed" conformation following agonist binding. In the present study, we have created a Zn 2؉ binding site in mGluR1b by mutating the residue Lys 260 to a histidine. Zinc acts as a noncompetitive antagonist of agonist-induced IP accumulation on the K260H mutant with an IC 50 value of 2 M. Alanine mutations of three potential "zinc coligands" in proximity to the introduced histidine in K260H knock out the ability of Zn 2؉ to antagonize the agonist-induced response. Zn 2؉ binding to K260H does not appear to affect the dimerization of the receptor. Instead, we propose that binding of zinc has introduced a structural constraint in the ATD lobe, preventing the formation of a "closed" conformation, and thus stabilizing a more or less inactive "open" form of the ATD. This study presents the first metal ion site constructed in a family C GPCR. Furthermore, it is the first time a metal ion site has been created in a region outside of the seven transmembrane regions of a GPCR and the loops connecting these. The findings offer valuable insight into the mechanism of ATD closure and family C receptor activation. Furthermore, the findings demonstrate that ATD regions other than those participating in agonist binding could be potential targets for new generations of ligands for this family of receptors.

“…Additionally, the exploration of 1,8-di-substituted-10-anthracene sulfonamide-based fluorophores, as suggested by our modeling studies, may yield a probe molecule with fluorescence wavelength properties optimized for use in fiber optic arrays. Future studies will focus on the structure-assisted redesign of both the fluorophore and CAII Ippolito and Christianson, 1994;Kiefer and Fierke, 1994;Ippolito et al, 1995;Kiefer et al, 1995;Lesburg and Christianson, 1995;Nair et al, 1995) in the optimization of a CAII-based zinc biosensor.…”

Section: Figmentioning

confidence: 99%

Unexpected Binding Mode of the Sulfonamide Fluorophore 5-Dimethylamino-1-naphthalene Sulfonamide to Human Carbonic Anhydrase II

Nair¹,

Elbaum²,

Christianson³

1996

The three-dimensional structure of human carbonic anhydrase II (CAII) complexed with the sulfonamide fluorophore 5-dimethylamino-1-naphthalene sulfonamide (dansylamide) has been determined to 2.1-Å resolution by x-ray crystallographic methods. Unlike other arylsulfonamide inhibitors of CAII, the naphthyl ring of dansylamide binds in a hydrophobic pocket in the active site, making van der Waals contacts with Val-121, Phe-131, Val-143, Leu-198, and Trp-209. Interestingly, a conformational change of Leu-198 is required to accommodate dansylamide binding, which rationalizes the enhanced dansylamide affinity measured for certain Sensors based upon biological macromolecules are increasingly used for the selective detection of numerous chemical entities, including the detection of trace quantities of metal ions. The advantages of metalloprotein-based biosensors over classical fluorometric chemical indicators for such applications include greater recognition and affinity for the target metal ion, exquisite discrimination among transition metals, and kinetically rapid biosensor-analyte association and dissociation. Recently, Thompson and Jones (1993) have exploited the selective recognition of zinc by the metalloenzyme carbonic anhydrase II (CAII) 1 in the design of an enzyme-based zinc biosensor. The physical basis of this sensor is the binding of zinc to the CAII apoenzyme, followed by binding of the fluorophore inhibitor 5-dimethylamino-1-naphthalene sulfonamide (dansylamide, K d ϭ 0.93 M (Nair et al., 1995); see Fig. 1) to the zinccontaining CAII holoenzyme. This leads to a titratable change in the fluorescence emission wavelength and intensity (Chen and Kernohan, 1967); nanomolar concentrations of zinc can be detected and quantified by ratiometric methods by measuring the emission of free dansylamide at 580 nm and the emission of bound dansylamide at 470 nm when excited at 290 nm.The zinc ion of CAII resides at the base of a 15-Å deep cleft, where it is liganded by the imidazole side chains of His-94, His-96, and His-119 (Håkansson et al., 1992). Hydroxide ion, which is the catalytic nucleophile in the CO 2 hydration mechanism, completes a tetrahedral metal coordination polyhedron. Zinc-bound hydroxide donates a hydrogen bond to the hydroxyl group of Thr-199, which in turn donates a hydrogen bond to Glu-106 (Eriksson et al., 1986, 1988aHåkansson et al., 1992). The binding of sulfonamide inhibitors displaces zinc-bound hydroxide and maintains the tetrahedral metal coordination polyhedron by the coordination of an ionized sulfonamide NH group, which also maintains the hydrogen bond interaction with Thr-199 (e.g. see Eriksson et al. (1988b) and Vidgren et al.