Hemozymes Peroxidase Activity Of Artificial Hemoproteins Constructed From the Streptomyces lividans Xylanase A and Iron(III)-Carboxy-Substituted Porphyrins

Abstract

To develop artificial hemoproteins that could lead to new selective oxidation biocatalysts, a strategy based on the insertion of various iron-porphyrin cofactors into Xylanase A (Xln10A) was chosen. This protein has a globally positive charge and a wide enough active site to accommodate metalloporphyrins that possess negatively charged substituents such as microperoxidase 8 (MP8), iron(III)-tetra-alpha4-ortho-carboxyphenylporphyrin (Fe(ToCPP)), and iron(III)-tetra-para-carboxyphenylporphyrin (Fe(TpCPP)). Coordination chemistry of the iron atom and molecular modeling studies showed that only Fe(TpCPP) was able to insert deeply into Xln10A, with a KD value of about 0.5 microM. Accordingly, Fe(TpCPP)-Xln10A bound only one imidazole molecule, whereas Fe(TpCPP) free in solution was able to bind two, and the UV-visible spectrum of the Fe(TpCPP)-Xln10A-imidazole complex suggested the binding of an amino acid of the protein on the iron atom, trans to the imidazole. Fe(TpCPP)-Xln10A was found to have peroxidase activity, as it was able to catalyze the oxidation of typical peroxidase cosubstrates such as guaiacol and o-dianisidine by H2O2. With these two cosubstrates, the KM value measured with the Fe(TpCPP)-Xln10A complex was higher than those values observed with free Fe(TpCPP), probably because of the steric hindrance and the increased hydrophobicity caused by the protein around the iron atom of the porphyrin. The peroxidase activity was inhibited by imidazole, and a study of the pH dependence of the oxidation of o-dianisidine suggested that an amino acid with a pKA of around 7.5 was participating in the catalysis. Finally, a very interesting protective effect against oxidative degradation of the porphyrin was provided by the protein.

Photoassisted Oxidation of Sulfides Catalyzed by Artificial Metalloenzymes Using Water as an Oxygen Source

“…A double reciprocal plot of the residual fluorescence intensity at 340 nm as a function of the 1-Mn final concentration allowed calculation of a K D value of 1.5 µM for 1-Mn-Xln10A and determined that only one ligand was bound per protein [18]. This K D value was similar to that already reported for the Fe(TpCPP)-Xln10A complex (K D = 0.5 µM) [21], and was in agreement with the fact that the binding of the porphyrin cofactor occurred thanks to interactions of three of its carboxylate substituents with amino-acid side chains of the protein and that no amino-acid side chain was interacting with the metal cation. This could be explained after molecular modeling calculations [18].…”

“…After lyophylization, 1-Mn was obtained in a quantitative yield and characterized by ESI-MS and UV-vis spectroscopy, in agreement with those already reported [18] and with an Mn(TpCPP) structure (Figure 1). To prepare the 1-Mn-Xln10A artificial hemoprotein, xylanase 10A (Xln10A) was first purified from the supernatant of S. lividans culture as reported earlier [21]. Solutions of the artificial hemoprotein were then prepared by incubating Xln10A in 50 mM sodium phosphate buffer, pH 7.0 with various amounts of 1-Mn for 30 min.…”

“…99 mM in 50 mM phosphate buffer solution (pH 7.0) was first assayed in the presence of 0.42 mM [Ru II (bpy)3] 2+ (13 eq.) as a photosensitizing agent, 12 mM To prepare the 1-Mn-Xln10A artificial hemoprotein, xylanase 10A (Xln10A) was first purified from the supernatant of S. lividans culture as reported earlier [21]. Solutions of the artificial hemoprotein were then prepared by incubating Xln10A in 50 mM sodium phosphate buffer, pH 7.0 with various amounts of 1-Mn for 30 min.…”

Incorporation of Manganese Complexes into Xylanase: New Artificial Metalloenzymes for Enantioselective Epoxidation

“…Recently, new artificial peroxidases have been created by association of Fe III -meso-tetrakis(p-carboxyphenyl)porphyrin-Fe-(TpCPP)-with xylanase 10A from Streptomyces lividans (Xln10A), a glycoside hydrolase that hydrolyses b-1,4 bonds in the main chain of xylan and is available at low cost and in large quantities. [25] Two further arguments also led us to choose xylanase A. Firstly, it was recently found that a synthetic iron(II) porphyrin could be incorporated into recombinant Thermotoga maritima xylanase B, producing a heat-resistant artificial haemoprotein that, like haemoglobin and myoglobin, can bind and release dioxygen in aqueous solution. [26] Secondly, the catalytic site of xylanase 10A globally appears as a crevice that is rich in positively charged amino acids capable of incorporating anionic porphyrins such as Fe(TpCPP).…”

“…In addition, it was shown that the Fe(TpCPP)-Xln10A complex no longer retained any glycosidic activity, which constituted an additional strong argument that the Fe(TpCPP) molecule was competitively incorporated into the substrate binding cleft of the (b/a) 8 TIM barrel and affected the enzymatic activity of the host xylanase. The protein thus produces steric hindrance around one of the faces of the Fe(TpCPP), [25] which constitutes an essential factor for inducing selectivity into the reactions catalysed by the porphyrin. The Xln10A-Fe-(TpCPP) artificial metalloprotein possessed a good peroxidase activity and was able to catalyse the oxidation of sulfides into sulfoxides by H 2 O 2 with high yields (up to 83 %) and with enantiomeric excesses of up to 40 %.…”