Discovery of a new family of carbonic anhydrases in the malaria pathogen Plasmodium falciparum —The η-carbonic anhydrases

Journal of Enzyme Inhibition and Medicinal Chemistry

et al. 2015

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Malassezia yeasts are almost exclusively the single eukaryotic members of the fungal flora of the skin. Malassezia globosa and Malassezia restricta are found on the skin of practically all humans. Malassezia globosa is highly implicated in the pathogenesis of dandruff and its genome encodes for only one carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the b-class (MgCA). It has been indeed demonstrated that in many pathogenic microorganisms, CAs are essential for their life cycle and their inhibition can lead to growth impairment and defects. In the previous work, the recombinant MgCA was investigated for its inhibition profile with sulfonamides, which in models of dandruff infection were able to protect animals from the fungal infection, allowing us to propose this enzyme as a new antidandruff target. MgCA was cloned as GST-fusion protein, but the yield was rather low and the protein was often found in inclusion bodies. Here, we propose an alternative procedure consisting in cloning the recombinant MgCA as His-Tag fusion protein. This procedure resulted in a good method to express and purify the active recombinant MgCA, and the protein recovery was better with respect to that used for preparing MG-CA (b-CA cloned as GST-fusion protein).

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A new procedure for the cloning, expression and purification of the β-carbonic anhydrase from the pathogenic yeastMalassezia globosa, an anti-dandruff drug target

Prete

Luca

Journal of Enzyme Inhibition and Medicinal Chemistry

et al. 2015

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“…4,5,[30][31][32][33][34][35][36][37][38] These enzymes are involved in a multitude of physiologic processes in organisms all over the phylogenetic tree, with six genetically distinct CA classes known to date: the a-, b-, c-, d-, f-and g-CAs. 26,[39][40][41][42][43][44][45][46][47][48][49][50] Their biochemical features are known in detail for at least four classes, together with their distribution and role in various organisms. 32,33,41,[48][49][50][51][52][53][54][55][56][57][58][59][60][61][62] Inhibition and activation studies of many such enzymes from vertebrates, protozoa, fungi and bacteria have shown that they are drug targets for obtaining pharmacological agents of the diuretic, antiglaucoma, antiobesity, antiepileptic, anticancer or anti-infective type.…”

mentioning

confidence: 99%

Sulfonamide inhibition studies of the γ-carbonic anhydrase from the Antarctic bacterium Pseudoalteromonas haloplanktis

Bioorganic & Medicinal Chemistry Letters

Luca

Prete

et al. 2015

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a b s t r a c tThe Antarctic bacterium Pseudoalteromonas haloplanktis encodes for a c-class carbonic anhydrase (CA, EC 4.2.1.1), which was cloned, purified and characterized. The enzyme (PhaCAc) has a good catalytic activity for the physiologic reaction of CO 2 hydration to bicarbonate and protons, with a k cat of 1.4 Â 10 5 s À1 and a k cat /K m of 1.9 Â 10 6 M À1 Â s À1 . A series of sulfonamides and a sulfamate were investigated as inhibitors of the new enzyme. Methazolamide and indisulam showed the best inhibitory properties (K I s of 86.7-94.7 nM). This contribution shed new light on c-CAs inhibition profiles with a relevant class of pharmacologic agents.Ó 2015 Elsevier Ltd. All rights reserved.Marine psychrophiles act as processors of the polar marine primary productivity constituting the base for the entire polar food web and, ultimately, feeding krill, fish, whales, penguins, and seabirds. 1,2 They play, in fact, a significant role in the so called 'substance turnover'. Moreover, a feature common to all psychrophiles are their remarkable ability to thrive under extremely cold and salty conditions. 3 Cold-adapted organisms have developed a number of adjustments at the molecular level to maintain metabolic functions at low temperatures, such as the production of enzymes, note as 'cold-enzyme'. 4-11 These enzymes are characterized by a specific activity at low and moderate temperatures higher than their mesophilic counterparts over a temperature range roughly covering 0-30°C and by a relative instability. 6,7,[11][12][13] Probably, in the case of psychrophilic microorganisms the selective pressure is essentially exerted towards the specific activity and not towards stability factors as happens in mesophilic or in thermophilic enzymes. The molecular structure of a 'cold-enzyme' is primarily characterized by an adequate plasticity of the molecule at the environmental temperature in order to accommodate the substrates with a minimum of energy expenditure. 14 'Cold-enzymes' naturally achieved a good compromise between activity and stability. There is a continuum in the adaptation of a protein to its environment. [4][5][6][7][8][9][10][11]13,[15][16][17][18][19][20][21][22] In fact, all known structural factors and weak interactions involved in protein stability are either reduced in number or modified in psychrophilic enzymes in order to increase their flexibility; but the same structural factors are also implicate for increasing the stability of the thermophilic proteins. [23][24][25][26][27][28][29] Carbonic anhydrases (CAs; EC 4.2.1.1) are metalloenzymes that catalyze CO 2 hydration to bicarbonate and protons. 4,5,[30][31][32][33][34][35][36][37][38] These enzymes are involved in a multitude of physiologic processes in organisms all over the phylogenetic tree, with six genetically distinct CA classes known to date: the a-, b-, c-, d-, f-and g-CAs. 26,[39][40][41][42][43][44][45][46][47][48][49][50] Their biochemical features are known in detail for at least four classes, together with their distribution and ...

“…Among them, the a-, b-, g-and d-CAs are broadly distributed in many microorganisms 1 , whereas the Z-CAs were described only in protozoa for the moment 5 . To date, two different b-CAs have been reported in Escherichia coli, the most investigated bacterium for the molecular biology viewpoint.…”

mentioning

confidence: 99%

Cloning, expression and biochemical characterization of aβ-carbonic anhydrase from the soil bacteriumEnterobactersp. B13

Eminoğlu

Journal of Enzyme Inhibition and Medicinal Chemistry

Aşık

et al. 2015

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A recombinant carbonic anhydrase (CA, EC 4.2.1.1) from the soil-dwelling bacterium Enterobacter sp. B13 was cloned and purified by Co 2+ affinity chromatography. Bioinformatic analysis showed that the new enzyme (denominated here B13-CA) belongs to the b-class CAs and to possess 95% homology with the ortholog enzyme from Escherichia coli encoded by the can gene, whereas its sequence homology with the other such enzyme from E. coli (encoded by the cynT gene) was of 33%. B13-CA was characterized kinetically as a catalyst for carbon dioxide hydration to bicarbonate and protons. The enzyme shows a significant catalytic activity, with the following kinetic parameters at 20 C and pH of 8.3: k cat of 4.8 Â 10 5 s À1 and k cat /K m of 5.6 Â 10 7 M À1 Â s À1. This activity was potently inhibited by acetazolamide which showed a K I of 78.9 nM. Although only this compound was investigated for the moment as B13-CA inhibitor, further studies may reveal new classes of inhibitors/activators of this enzyme which may show biomedical or environmental applications, considering the posssible role of this enzyme in CaCO 3 biomineralization processes.