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  • br Phylogenetic analysis KSTDs have rather

    2019-10-09


    Phylogenetic analysis — Δ1-KSTDs have rather diverse amino Angiotensin Fragment 1-7 acetate clinical sequences. A sequence distance analysis using the program MEGA6 [75] of all currently biochemically characterized Δ1-KSTDs yielded a largest p-distance [76] of 0.67 (on a scale of 0-1) for the Δ1-KSTDs from the actinobacteria Nocardioides simplex IFO 12069 (GenPept BAA07186.1; also called Pimelobacter simplex, Arthrobacter simplex, or Corynebacterium simplex) [77] and M. neoaurum ATCC 25795 (GenPept AHG53938.1) [62] with an amino acid identity of 30%. When all currently available putative Δ1-KSTD sequences are included in the analysis, a p-distance as large as 0.72 was found for the Δ1-KSTDs from the actinobacterium Aeromicrobium marinum (GenPept WP_007078704.1) and the proteobacterium Vitreoscilla stercoraria (GenPept WP_040755675.1), which have a sequence identity of 26% only. A phylogenetic analysis of Δ1-KSTD sequences resulted in a cladogram with several different clades, i.e. clades A, B, C, and D (Fig. 3). All Δ1-KSTDs from fungi of the phylum Ascomycota and bacteria of the phylum Chloroflexi are clustered in subclade A1, which also includes an archaeal Δ1-KSTD from Candidatus Caldiarchaeum subterraneum. Subclade A2 contains actinobacterial Δ1-KSTDs in one cluster and proteobacterial enzymes in the other cluster. Subclade B1 mostly contains Δ1-KSTDs from Firmicutes bacteria and the amoebozoa P. pallidum PN500. Subclade B2 is mostly occupied by actinobacterial enzymes, but it also comprises a Δ1-KSTD from the bacterium Empedobacter falsenii, a member of the phylum Bacteroidetes. Although Δ1-KSTDs from Actinobacteria can be found in virtually all clades, the majority of these enzymes are in subclade B2 and in clade C. Similarly, the enzymes from Proteobacteria are present in several clades, but mainly clustered in clade D. This latter clade also accommodates some actinobacterial Δ1-KSTDs. Hence, in general, Δ1-KSTDs are phylogenetically clustered on the basis of their microbial sources. The phylogenetic tree and substrate specificity — The phylogenetic analysis placed Δ1-KSTDs with similar substrate specificities in the same clade. For instance, Δ1-KSTD3 from R. erythropolis SQ1 (GenPept ABW74859.1) and Δ1-KSTD from M. tuberculosis H37Rv (GenPept NP_218054.1), which are both active on 5α-3-ketosteroids, but not on Δ4-3-ketosteroids [28], are both in subclade B2. The subclade B2 Δ1-KSTD from M. neoaurum ATCC 25795 (GenPept ACV13200.1) is also active on 5α-3-ketosteroids (5α-testosterone (23)), although this enzyme has a more relaxed substrate specificity, and can also convert Δ4-3-ketosteroid substrates [62]. In contrast, the clade C Δ1-KSTD1 of R. erythropolis SQ1 (GenPept AAF19054.1) is active on Δ4-3-ketosteroids, while the subclade B2 Δ1-KSTD3 of the same bacterial strain (GenPept ABW74859.1) prefers 5α-3-ketosteroids [28]. Three Δ1-KSTD isoenzymes from M. neoaurum ATCC 25795, assigned to subclade B1 (GenPept AHG53938.1), subclade B2 (GenPept ACV13200.1), and clade C (GenPept AHG53939.1), were also reported to display significant differences in substrate preference [62]. Thus, these observations suggest that Δ1-KSTDs residing in the same clade have similar substrate specificities, which may differ from the substrate specificities of Δ1-KSTDs from other clades. Distribution of isoenzymes in the phylogenetic tree — In the cladogram, multiple Δ1-KSTD isoenzymes of a particular organism tend to be distributed across several clades, instead of clustered in a single clade. For instance, the five Δ1-KSTD isoenzymes from the actinobacterium R. opacus PD630 appear in clades B (subclades B1 and B2), C, and D. Similarly, the Δ1-KSTD isoenzymes from the actinobacteria R. erythropolis, N. simplex, and M. neoaurum, as well as the proteobacterium Novosphingobium malaysiense are found in several different clades. If the presence in different clades is correlated with differences in substrate specificity, as suggested above, these distributions may reflect the capability of the corresponding microorganisms to use a diverse variety of steroid substrates.