Supporting information to the Figure 4

Phylogenetic relationships of 47 collection strains harboring erm resistance genes. An alignment of 16S rRNA gene sequences. A phylogram constructed by neighbor-joining method based on Jukes-Cantor distance matrices calculated from 1000 bootstrap replicates of the alignment

Supporting infomation to the Figure 2.

Phylogenetic analysis of 17 putative ABC transporters from the isolated strains with 23 reference sequences of ABC transporters described in actinobacteria. An alignment of amino-acid ABC transporter sequences. The phylogenies inferred by Bayesian analysis, maximum-likelihood and neighbor-joining methods

Lincomycin Biosynthesis Involves a Tyrosine Hydroxylating Heme Protein of an Unusual Enzyme Family

<div><p>The gene lmbB2 of the lincomycin biosynthetic gene cluster of <i>Streptomyces lincolnensis</i> ATCC 25466 was shown to code for an unusual tyrosine hydroxylating enzyme involved in the biosynthetic pathway of this clinically important antibiotic. LmbB2 was expressed in <i>Escherichia coli</i>, purified near to homogeneity and shown to convert tyrosine to 3,4-dihydroxyphenylalanine (DOPA). In contrast to the well-known tyrosine hydroxylases (EC 1.14.16.2) and tyrosinases (EC 1.14.18.1), LmbB2 was identified as a heme protein. Mass spectrometry and Soret band-excited Raman spectroscopy of LmbB2 showed that LmbB2 contains heme b as prosthetic group. The CO-reduced differential absorption spectra of LmbB2 showed that the coordination of Fe was different from that of cytochrome P450 enzymes. LmbB2 exhibits sequence similarity to Orf13 of the anthramycin biosynthetic gene cluster, which has recently been classified as a heme peroxidase. Tyrosine hydroxylating activity of LmbB2 yielding DOPA in the presence of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH₄) was also observed. Reaction mechanism of this unique heme peroxidases family is discussed. Also, tyrosine hydroxylation was confirmed as the first step of the amino acid branch of the lincomycin biosynthesis.</p></div

Spectrometric analysis of MBP2*- LmbB2.

<p>Absorption spectra (A) of oxidized (dashed line) and reduced (full line) forms of MBP2*- LmbB2 bore witness of heme presence in the MBP2*- LmbB2 molecule showing Soret band at 404 nm and its shift under dithionite treatment. Reduced CO-differential spectrum (B) of the MBP2*- LmbB2 did not show a maximum at 450 nm indicating that LmbB2 does not belong to the cytochrome P450 superfamily.</p

Resonance Raman spectrum of MBP2*- LmbB2.

<p>MBP2*- LmbB2 (trace A) was superimposed on a fluorescence background. Background corrected spectrum (trace B) was expanded 25× to highlight details. Upper part of the background-corrected spectrum exhibited pattern typical for the Soret band-excited RRS spectra of various heme-containing proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079974#pone.0079974-Spiro1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079974#pone.0079974-Rousseau1" target="_blank">[22]</a>.</p

Analysis of LmbB2 by CD spectroscopy.

<p>(A) The far-UV CD spectrum of LmbB2. (B) Thermal denaturation of LmbB2 in the presence of 0 µM (blue curve), 50 µM (red curve) and 250 µM (green curve) concentration of BH₄ bear witness of the stabilization of the LmbB2 structure by BH₄.</p

Effect of BH₄ on melting temperature (<i>T</i>_m) of LmbB2.

<p>Effect of BH₄ on melting temperature (<i>T</i>_m) of LmbB2.</p

Effect of metal chelators, detergents, electron donors on the LmbB2 activity.

*<p>compounds were tested in concentrations ranging from 2 µm to 2 mM. The results were consistent with the conclusion that BH₄ and NADH increase and decrease, respectively, the LmbB2 activity.</p