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Identification of a novel Baeyer-Villiger monooxygenase from Acinetobacter radioresistens: close relationship to the Mycobacterium tuberculosis prodrug activator EtaA.

Minerdi D, Zgrablic I, Sadeghi SJ, Gilardi G - Microb Biotechnol (2012)

Bottom Line: Phylogenetic analysis placed the sequence of this novel BVMO in the same clade of the prodrug activator ethionamide monooxygenase (EtaA) and it bears only a distant relation to the other known class I BVMO proteins.In silico analysis of the 3D model of the S13 BVMO generated by homology modelling also supports the similarities with EtaA by binding ethionamide to the active site.In vitro experiments carried out with the purified enzyme confirm that this novel BVMO is indeed capable of typical Baeyer-Villiger reactions as well as oxidation of the prodrug ethionamide.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences and Systems Biology, University of Torino, via Accademia Albertina 13, 10123 Torino, Italy.

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Multiple sequence alignment of predicted amino acid sequences corresponding to alkane monooxygenase genes from various bacteria. The three conserved histidine boxes (Hist‐1, Hist‐2 and Hist‐3) are highlighted in red and the HYG‐motif is highlighted in violet. The amino acids are indicated by single‐letter codes. Gaps were introduced for optimal alignment. Identical residues between two sequences are indicated by an asterisk (*); similar residues are indicated by one dot (low similarity) or two dots (high similarity). Conserved histidine of the fingerprint motif are written in bold letters. Gordonia = alkane 1‐monooxygenase from Gordonia sp. TF6, BAD67020.1; Rhodococcus = alkane 1‐monooxygenase from Rhodococcus ruber, ACX30755.1; Mycobacterium = alkane 1‐monooxygenase from Mycobacterium sp. Spyr 1, YP_004078475.1; Streptomyces = alkane 1‐monooxygenase from Streptomyces sp. AA4, ZP_07282765.1; Thermonospora = alkane 1‐monooxygenase from Thermonospora curvata DSM 43183, YP_003298195.1; Ralstonia = alkane 1‐monooxygenase from Ralstonia sp., ZP_07673680.1; Burkholderia = alkane 1‐monooxygenase from Burkholderia phytofirmans YP_001889129; Alcanivorax = alkane 1‐monooxygenase from Alcanivorax dieselolei, AAT91722.2; SH164 = alkane hydroxylase B from Acinetobacter radioresistens SH164, ZP_06072466.1; SK 82 = alkane 1‐monooxygenase from Acinetobacter radioresistens SK82, ZP_05361594.1; Ajunii = alkane hydroxylase B from Acinetobacter junii, ZP_06066074.1; Acalcoaceticus = alkane 1‐monooxygenase from Acinetobacter calcoaceticus, CAB51020.1; Abaumannii = alkane 1‐monooxgenase from Acinetobacter baumannii, YP_001707231.1; Ahaemolyticus = alkane hydroxylase from Acinetobacter haemolyticus, AAS93604.4; Psychrobacter = alkane 1‐monooxygenase from Psychrobacter sp. PRwf‐1, YP_001280943.1; S13 = alkane 1‐monooxygenase from Acinetobacter radioresistens S13, GU145276 (this work).
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f4: Multiple sequence alignment of predicted amino acid sequences corresponding to alkane monooxygenase genes from various bacteria. The three conserved histidine boxes (Hist‐1, Hist‐2 and Hist‐3) are highlighted in red and the HYG‐motif is highlighted in violet. The amino acids are indicated by single‐letter codes. Gaps were introduced for optimal alignment. Identical residues between two sequences are indicated by an asterisk (*); similar residues are indicated by one dot (low similarity) or two dots (high similarity). Conserved histidine of the fingerprint motif are written in bold letters. Gordonia = alkane 1‐monooxygenase from Gordonia sp. TF6, BAD67020.1; Rhodococcus = alkane 1‐monooxygenase from Rhodococcus ruber, ACX30755.1; Mycobacterium = alkane 1‐monooxygenase from Mycobacterium sp. Spyr 1, YP_004078475.1; Streptomyces = alkane 1‐monooxygenase from Streptomyces sp. AA4, ZP_07282765.1; Thermonospora = alkane 1‐monooxygenase from Thermonospora curvata DSM 43183, YP_003298195.1; Ralstonia = alkane 1‐monooxygenase from Ralstonia sp., ZP_07673680.1; Burkholderia = alkane 1‐monooxygenase from Burkholderia phytofirmans YP_001889129; Alcanivorax = alkane 1‐monooxygenase from Alcanivorax dieselolei, AAT91722.2; SH164 = alkane hydroxylase B from Acinetobacter radioresistens SH164, ZP_06072466.1; SK 82 = alkane 1‐monooxygenase from Acinetobacter radioresistens SK82, ZP_05361594.1; Ajunii = alkane hydroxylase B from Acinetobacter junii, ZP_06066074.1; Acalcoaceticus = alkane 1‐monooxygenase from Acinetobacter calcoaceticus, CAB51020.1; Abaumannii = alkane 1‐monooxgenase from Acinetobacter baumannii, YP_001707231.1; Ahaemolyticus = alkane hydroxylase from Acinetobacter haemolyticus, AAS93604.4; Psychrobacter = alkane 1‐monooxygenase from Psychrobacter sp. PRwf‐1, YP_001280943.1; S13 = alkane 1‐monooxygenase from Acinetobacter radioresistens S13, GU145276 (this work).

Mentions: The AlkB protein of A. radioresistens S13 possess eight histidines that are highly conserved in non‐haem iron integral membrane alkane hydroxylases and desaturases, and that are believed to be required for catalytic activity by these enzymes (Shanklin et al., 1994) (Fig. > 4). Sequences corresponding to the three histidine boxes Hist1 = HE(L/M)XHK, Hist2 = EHXXGHH and Hist3 = LQRH(S/A)DHHA are highly conserved in all bacterial alkane monooxygenases (Fig. 4). The Hist3 box is the longest and almost perfectly conserved sequence in all alkane hydroxylases, but is not well conserved in other closely related hydrocarbon monooxygenases. An additional well‐conserved histidine box NYXEHYG(L/M) containing the HYG motif that is located about 30 amino acids upstream of the Hist3 box is also present (Fig. 4) (Smits et al., 1999). This HYG motif is also well conserved in related hydrocarbon monooxygenases.


Identification of a novel Baeyer-Villiger monooxygenase from Acinetobacter radioresistens: close relationship to the Mycobacterium tuberculosis prodrug activator EtaA.

Minerdi D, Zgrablic I, Sadeghi SJ, Gilardi G - Microb Biotechnol (2012)

Multiple sequence alignment of predicted amino acid sequences corresponding to alkane monooxygenase genes from various bacteria. The three conserved histidine boxes (Hist‐1, Hist‐2 and Hist‐3) are highlighted in red and the HYG‐motif is highlighted in violet. The amino acids are indicated by single‐letter codes. Gaps were introduced for optimal alignment. Identical residues between two sequences are indicated by an asterisk (*); similar residues are indicated by one dot (low similarity) or two dots (high similarity). Conserved histidine of the fingerprint motif are written in bold letters. Gordonia = alkane 1‐monooxygenase from Gordonia sp. TF6, BAD67020.1; Rhodococcus = alkane 1‐monooxygenase from Rhodococcus ruber, ACX30755.1; Mycobacterium = alkane 1‐monooxygenase from Mycobacterium sp. Spyr 1, YP_004078475.1; Streptomyces = alkane 1‐monooxygenase from Streptomyces sp. AA4, ZP_07282765.1; Thermonospora = alkane 1‐monooxygenase from Thermonospora curvata DSM 43183, YP_003298195.1; Ralstonia = alkane 1‐monooxygenase from Ralstonia sp., ZP_07673680.1; Burkholderia = alkane 1‐monooxygenase from Burkholderia phytofirmans YP_001889129; Alcanivorax = alkane 1‐monooxygenase from Alcanivorax dieselolei, AAT91722.2; SH164 = alkane hydroxylase B from Acinetobacter radioresistens SH164, ZP_06072466.1; SK 82 = alkane 1‐monooxygenase from Acinetobacter radioresistens SK82, ZP_05361594.1; Ajunii = alkane hydroxylase B from Acinetobacter junii, ZP_06066074.1; Acalcoaceticus = alkane 1‐monooxygenase from Acinetobacter calcoaceticus, CAB51020.1; Abaumannii = alkane 1‐monooxgenase from Acinetobacter baumannii, YP_001707231.1; Ahaemolyticus = alkane hydroxylase from Acinetobacter haemolyticus, AAS93604.4; Psychrobacter = alkane 1‐monooxygenase from Psychrobacter sp. PRwf‐1, YP_001280943.1; S13 = alkane 1‐monooxygenase from Acinetobacter radioresistens S13, GU145276 (this work).
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f4: Multiple sequence alignment of predicted amino acid sequences corresponding to alkane monooxygenase genes from various bacteria. The three conserved histidine boxes (Hist‐1, Hist‐2 and Hist‐3) are highlighted in red and the HYG‐motif is highlighted in violet. The amino acids are indicated by single‐letter codes. Gaps were introduced for optimal alignment. Identical residues between two sequences are indicated by an asterisk (*); similar residues are indicated by one dot (low similarity) or two dots (high similarity). Conserved histidine of the fingerprint motif are written in bold letters. Gordonia = alkane 1‐monooxygenase from Gordonia sp. TF6, BAD67020.1; Rhodococcus = alkane 1‐monooxygenase from Rhodococcus ruber, ACX30755.1; Mycobacterium = alkane 1‐monooxygenase from Mycobacterium sp. Spyr 1, YP_004078475.1; Streptomyces = alkane 1‐monooxygenase from Streptomyces sp. AA4, ZP_07282765.1; Thermonospora = alkane 1‐monooxygenase from Thermonospora curvata DSM 43183, YP_003298195.1; Ralstonia = alkane 1‐monooxygenase from Ralstonia sp., ZP_07673680.1; Burkholderia = alkane 1‐monooxygenase from Burkholderia phytofirmans YP_001889129; Alcanivorax = alkane 1‐monooxygenase from Alcanivorax dieselolei, AAT91722.2; SH164 = alkane hydroxylase B from Acinetobacter radioresistens SH164, ZP_06072466.1; SK 82 = alkane 1‐monooxygenase from Acinetobacter radioresistens SK82, ZP_05361594.1; Ajunii = alkane hydroxylase B from Acinetobacter junii, ZP_06066074.1; Acalcoaceticus = alkane 1‐monooxygenase from Acinetobacter calcoaceticus, CAB51020.1; Abaumannii = alkane 1‐monooxgenase from Acinetobacter baumannii, YP_001707231.1; Ahaemolyticus = alkane hydroxylase from Acinetobacter haemolyticus, AAS93604.4; Psychrobacter = alkane 1‐monooxygenase from Psychrobacter sp. PRwf‐1, YP_001280943.1; S13 = alkane 1‐monooxygenase from Acinetobacter radioresistens S13, GU145276 (this work).
Mentions: The AlkB protein of A. radioresistens S13 possess eight histidines that are highly conserved in non‐haem iron integral membrane alkane hydroxylases and desaturases, and that are believed to be required for catalytic activity by these enzymes (Shanklin et al., 1994) (Fig. > 4). Sequences corresponding to the three histidine boxes Hist1 = HE(L/M)XHK, Hist2 = EHXXGHH and Hist3 = LQRH(S/A)DHHA are highly conserved in all bacterial alkane monooxygenases (Fig. 4). The Hist3 box is the longest and almost perfectly conserved sequence in all alkane hydroxylases, but is not well conserved in other closely related hydrocarbon monooxygenases. An additional well‐conserved histidine box NYXEHYG(L/M) containing the HYG motif that is located about 30 amino acids upstream of the Hist3 box is also present (Fig. 4) (Smits et al., 1999). This HYG motif is also well conserved in related hydrocarbon monooxygenases.

Bottom Line: Phylogenetic analysis placed the sequence of this novel BVMO in the same clade of the prodrug activator ethionamide monooxygenase (EtaA) and it bears only a distant relation to the other known class I BVMO proteins.In silico analysis of the 3D model of the S13 BVMO generated by homology modelling also supports the similarities with EtaA by binding ethionamide to the active site.In vitro experiments carried out with the purified enzyme confirm that this novel BVMO is indeed capable of typical Baeyer-Villiger reactions as well as oxidation of the prodrug ethionamide.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences and Systems Biology, University of Torino, via Accademia Albertina 13, 10123 Torino, Italy.

Show MeSH
Related in: MedlinePlus