Peptide-derived cyclophanes inhabit
a unique niche in the chemical
space of macrocyclic peptides with several examples of pharmaceutical
importance. Although both synthetic and biocatalytic methods are available
for constructing these macrocycles, versatile (bio)catalysts able
to incorporate a variety of amino acids that compose the macrocycle
would be useful for the creation of diverse peptide cyclophanes. In
this report, we synergized the use of bioinformatic tools to map the
biosynthetic landscape of radical SAM enzymes (3-CyFEs) that catalyze
three-residue cyclophane formation in the biosynthesis of a new family
of RiPP natural products, the triceptides. This analysis revealed
3940 (3113 unique) putative precursor sequences predicted to be modified
by 3-CyFEs. Several uncharacterized maturase systems were identified
that encode unique precursor types. Functional studies were carried
out in vivo in Escherichia coli to
identify modified precursors containing His and Tyr residues. NMR
analysis of the products revealed that Tyr and His can also be incorporated
into cyclophane macrocycles by 3-CyFEs. Collectively, all aromatic
amino acids can be incorporated by 3-CyFEs, and the cyclophane formation
strictly occurs via a C(sp2)–C(sp3) cross-link
between the (hetero)aromatic ring to Cβ. In addition to 3-CyFEs,
we functionally validated an Fe(II)/α-ketoglutarate-dependent
hydroxylase, resulting in β-hydroxylated residues within the
cyclophane rings. This study reveals the potential breadth of triceptide
precursors and a systematic approach for studying these enzymes to
broaden the diversity of peptide macrocycles.
A single Nitrospira sublineage I OTU was found
to perform nitrite oxidation in full-scale domestic wastewater treatment
plants (WWTPs) in the tropics. This taxon had an apparent oxygen affinity
constant lower than that of the full-scale domestic activated sludge
cohabitating ammonium oxidizing bacteria (AOB) (0.09 ± 0.02 g
O2 m–3 versus 0.3 ± 0.03 g O2 m–3). Thus, nitrite oxidizing bacteria
(NOB) may in fact thrive under conditions of low oxygen supply. Low
dissolved oxygen (DO) conditions selected for and high aeration inhibited
the NOB in a long-term lab-scale reactor. The relative abundance of Nitrospira sublineage I gradually decreased with increasing
DO until it was washed out. Nitritation was sustained even after the
DO was lowered subsequently. The morphologies of AOB and NOB microcolonies
responded to DO levels in accordance with their oxygen affinities.
NOB formed densely packed spherical clusters with a low surface area-to-volume
ratio compared to the Nitrosomonas-like AOB clusters,
which maintained a porous and nonspherical morphology. In conclusion,
the effect of oxygen on AOB/NOB population dynamics depends on which
OTU predominates given that oxygen affinities are species-specific,
and this should be elucidated when devising operating strategies to
achieve mainstream partial nitritation.
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