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Manteca A,
Pelaez AI,
Zardoya R,
Sanchez J,
( 2006 ) Actinobacteria cyclophilins: phylogenetic relationships and description of new class- and order-specific paralogues. PMID : 17103061 : DOI : 10.1007/s00239-005-0130-3 Abstract >>
Cyclophilins are folding helper enzymes belonging to the class of peptidyl-prolyl cis-trans isomerases (PPIases; EC 5.2.1.8) that catalyze the cis-trans isomerization of peptidyl-prolyl bonds in proteins. They are ubiquitous proteins present in almost all living organisms analyzed to date, with extremely rare exceptions. Few cyclophilins have been described in Actinobacteria, except for three reported in the genus Streptomyces and another one in Mycobacterium tuberculosis. In this study, we performed a complete phylogenetic analysis of all Actinobacteria cyclophilins available in sequence databases and new Streptomyces cyclophilin genes sequenced in our laboratory. Phylogenetic analyses of cyclophilins recovered six highly supported groups of paralogy. Streptomyces appears as the bacteria having the highest cyclophilin diversity, harboring proteins from four groups. The first group was named "A" and is made up of highly conserved cytosolic proteins of approximately 18 kDa present in all Actinobacteria. The second group, "B," includes cytosolic proteins widely distributed throughout the genus Streptomyces and closely related to eukaryotic cyclophilins. The third group, "M" cyclophilins, consists of high molecular mass cyclophilins (approximately 30 kDa) that contain putative membrane binding domains and would constitute the only membrane cyclophilins described to date in bacteria. The fourth group, named "C" cyclophilins, is made up of proteins of approximately 18 kDa that are orthologous to Gram-negative proteobacteria cyclophilins. Ancestral character reconstruction under parsimony was used to identify shared-derived (and likely functionally important) amino acid residues of each paralogue. Southern and Western blot experiments were performed to determine the taxonomic distribution of the different cyclophilins in Actinobacteria.
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2. |
Kim BJ,
Kim CJ,
Chun J,
Koh YH,
Lee SH,
Hyun JW,
Cha CY,
Kook YH,
( 2004 ) Phylogenetic analysis of the genera Streptomyces and Kitasatospora based on partial RNA polymerase beta-subunit gene (rpoB) sequences. PMID : 15023980 : DOI : 10.1099/ijs.0.02941-0 Abstract >>
The RNA polymerase beta-subunit genes (rpoB) of 67 Streptomyces strains, representing 57 species, five Kitasatospora strains and Micromonospora echinospora KCTC 9549 were partially sequenced using a pair of rpoB PCR primers. Among the streptomycetes, 99.7-100 % similarity within the same species and 90.2-99.3 % similarity at the interspecific level were observed by analysis of the determined rpoB sequences. The topology of the phylogenetic tree based on rpoB sequences was similar to that of 16S rDNA. The five Kitasatospora strains formed a stable monophyletic clade and a sister group to the clade comprising all Streptomyces species. Although there were several discrepancies in the details, considerable agreement was found between the results of rpoB analysis and those of numerical phenetic classification. This study demonstrates that analysis of rpoB can be used as an alternative genetic method in parallel to conventional taxonomic methods, including numerical phenetic and 16S rDNA analyses, for the phylogenetic analyses of the genera Streptomyces and Kitasatospora.
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3. |
Burks EA,
Yan W,
Johnson WH,
Li W,
Schroeder GK,
Min C,
Gerratana B,
Zhang Y,
Whitman CP,
( 2011 ) Kinetic, crystallographic, and mechanistic characterization of TomN: elucidation of a function for a 4-oxalocrotonate tautomerase homologue in the tomaymycin biosynthetic pathway. PMID : 21809870 : DOI : 10.1021/bi200947w PMC : PMC3164527 Abstract >>
The biosynthesis of the C ring of the antitumor antibiotic agent, tomaymycin, is proposed to proceed through five enzyme-catalyzed steps from l-tyrosine. The genes encoding these enzymes have recently been cloned and their functions tentatively assigned, but there is limited biochemical evidence supporting the assignments of the last three steps. One enzyme, TomN, shows 58% pairwise sequence similarity with 4-oxalocrotonate tautomerase (4-OT), an enzyme found in a catabolic pathway for aromatic hydrocarbons. The TomN sequence includes three amino acids (Pro-1, Arg-11, and Arg-39) that have been identified as critical catalytic residues in 4-OT. However, the proposed substrate for TomN is very different from that processed by 4-OT. To establish the function and mechanism of TomN and its relationship with 4-OT, we conducted kinetic, mutagenic, and structural studies. The kinetic parameters for TomN, and four alanine mutants, P1A, R11A, R39A, and R61A, were determined using 2-hydroxymuconate, the substrate for 4-OT. The TomN-catalyzed reaction using this substrate compares favorably to that of 4-OT. In addition, the kinetic parameters for the P1A, R11A, and R39A mutants of TomN parallel the trends observed for the corresponding 4-OT mutants, implicating an analogous mechanism. A high-resolution crystal structure (1.4 ?) of TomN shows that the overall structure and the active site region are highly similar to those of 4-OT with a root-mean-square deviation of 0.81 ?. Moreover, key active site residues are positionally conserved. The combined results suggest that the tentative assignment for TomN and the proposed sequence of events in the biosynthetic pathway leading to the formation of the C ring of tomaymycin might not be correct. An alternative pathway that awaits biochemical confirmation is proposed.
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4. |
Li W,
Chou S,
Khullar A,
Gerratana B,
( 2009 ) Cloning and characterization of the biosynthetic gene cluster for tomaymycin, an SJG-136 monomeric analog. PMID : 19270147 : DOI : 10.1128/AEM.02325-08 PMC : PMC2681672 Abstract >>
Tomaymycin produced by Streptomyces achromogenes is a naturally produced pyrrolobenzodiazepine (PBD). The biosynthetic gene cluster for tomaymycin was identified and sequenced. The gene cluster analysis reveals a novel biosynthetic pathway for the anthranilate moiety of PBDs. Gene replacement and chemical complementation studies were used to confirm the proposed biosynthetic pathway.
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5. |
( 1998 ) Cloning and expression of the ApaLI, NspI, NspHI, SacI, ScaI, and SapI restriction-modification systems in Escherichia coli. PMID : 9862476 : DOI : 10.1007/s004380050890 Abstract >>
The genes encoding the ApaLI (5'-GTGCAC-3'), NspI (5'-RCATGY-3'), NspHI (5'-RCATGY-3'), SacI (5'-GAGCTC-3'), SapI (5'-GCTCTTCN1-3', 5'-N4GAAGAGC-3') and ScaI (5'-AGTACT-3') restriction-modification systems have been cloned in E. coli. Amino acid sequence comparison of M.ApaLI, M.NspI, M.NspHI, and M.SacI with known methylases indicated that they contain the ten conserved motifs characteristic of C5 cytosine methylases. NspI and NspHI restriction-modification systems are highly homologous in amino acid sequence. The C-termini of the NspI and NlaIII (5'-CATG-3') restriction endonucleases share significant similarity. 5mC modification of the internal C in a SacI site renders it resistant to SacI digestion. External 5mC modification of a SacI site has no effect on SacI digestion. N4mC modification of the second base in the sequence 5'-GCTCTTC-3' blocks SapI digestion. N4mC modification of the other cytosines in the SapI site does not affect SapI digestion. N4mC modification of ScaI site blocks ScaI digetion. A DNA invertase homolog was found adjacent to the ApaLI restriction-modification system. A DNA transposase subunit homolog was found upstream of the SapI restriction endonuclease gene.
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