Tolyporphin—An Unusual Green Chlorin‐like Dioxobacteriochlorin

Hughes

et al. 2017

Phytochemical Analysis

Section: Introductionmentioning

confidence: 99%

Quantitation of Tolyporphins, Diverse Tetrapyrrole Secondary Metabolites with Chlorophyll‐Like Absorption, from a Filamentous Cyanobacterium–Microbial Community

Hughes

et al. 2017

Phytochemical Analysis

“…Tolyporphins A to J, L, and M are bacteriochlorins (containing two reduced pyrrole rings), whereas chlorophylls, the photosynthetic pigments of cyanobacteria, are chlorins and hence contain only one reduced ring. The presence of the two oxo groups in tolyporphin A results in spectral properties more resembling those of chlorophyll a rather than bacteriochlorophyll a (10,11), and yet the biosynthesis of bacteriochlorins generally is the province of anoxygenic photosynthetic bacteria, not cyanobacteria. Why bacteriochlorins are present in cyanobacteria is a mystery with possible evolutionary implications (3).…”

mentioning

confidence: 99%

Genome Sequence and Composition of a Tolyporphin-Producing Cyanobacterium-Microbial Community

Hughes

et al. 2017

Appl Environ Microbiol

The cyanobacterial culture HT-58-2 was originally described as a strain of with the ability to produce tolyporphins, which comprise a family of distinct tetrapyrrole macrocycles with reported efflux pump inhibition properties. Upon reviving the culture from what was thought to be a nonextant collection, studies of culture conditions, strain characterization, phylogeny, and genomics have been undertaken. Here, HT-58-2 was shown by 16S rRNA analysis to closely align with strains and not with isolates. Light, fluorescence, and scanning electron microscopy revealed cyanobacterium filaments that are decorated with attached bacteria and associated with free bacteria. Metagenomic surveys of HT-58-2 cultures revealed a diversity of bacteria dominated by, 97% of which are species. A dimethyl sulfoxide washing procedure was found to yield enriched cyanobacterial DNA (presumably by removing community bacteria) and sequence data sufficient for genome assembly. The finished, closed HT-58-2Cyano genome consists of 7.85 Mbp (42.6% G+C) and contains 6,581 genes. All genes for biosynthesis of tetrapyrroles (e.g., heme, chlorophyll, and phycocyanobilin) and almost all for cobalamin were identified dispersed throughout the chromosome. Among the 6,177 protein-encoding genes, coding sequences (CDSs) for all but two of the eight enzymes for conversion of glutamic acid to protoporphyrinogen IX also were found within one major gene cluster. The cluster also includes 10 putative genes (and one hypothetical gene) encoding proteins with domains for a glycosyltransferase, two cytochrome P450 enzymes, and a flavin adenine dinucleotide (FAD)-binding protein. The composition of the gene cluster suggests a possible role in tolyporphin biosynthesis. A worldwide search more than 25 years ago for cyanobacterial natural products with anticancer activity identified a culture (HT-58-2) from Micronesia that produces tolyporphins. Tolyporphins are tetrapyrroles, like chlorophylls, but have several profound structural differences that reside outside the bounds of known biosynthetic pathways. To begin probing the biosynthetic origin and biological function of tolyporphins, our research has focused on studying the cyanobacterial strain, about which almost nothing has been previously reported. We find that the HT-58-2 culture is composed of the cyanobacterium and a community of associated bacteria, complicating the question of which organisms make tolyporphins. Elucidation of the cyanobacterial genome revealed an intriguing gene cluster that contains tetrapyrrole biosynthesis genes and a collection of unknown genes, suggesting that the cluster may be responsible for tolyporphin production. Knowledge of the genome and the gene cluster sharply focuses research to identify related cyanobacterial producers of tolyporphins and delineate the tolyporphin biosynthetic pathway.

“…c, d, f), while rare events, are illustrative (1,2). The discovery of tolyporphin A in a cyanobacterial culture in Micronesia in the late 1980s by a team from the University of Hawaii (3) has also raised many such questions, particularly since the absorption spectrum resembles that of chlorophyll a, the compound is photoactive (4,5), the structure lies outside the known biosynthetic paradigm for tetrapyrroles (6,7), and the chromophore of tolyporphin A is a dioxobacteriochlorin yet the compound is found in a (chlorophyll-containing) cyanobacterium. A family of tolyporphins has subsequently been discovered, now including tolyporphins A-R (8)(9)(10)(11).…”

Section: Introductionmentioning

confidence: 99%

Fluorescence Assay for Tolyporphins Amidst Abundant Chlorophyll in Crude Cyanobacterial Extracts

Nguyen

Photochem & Photobiology

Taniguchi

et al. 2021

Tolyporphins are distinctive tetrapyrrole natural products found singularly in a filamentous cyanobacterial‐microbial holobiont (termed HT‐58‐2) from Micronesia. The absorption and fluorescence features of tolyporphins resemble those of chlorophyll a, complicating direct analysis of culture samples. Treatment of the crude (unfractionated) organic extract (CH2Cl2/2‐propanol, 1:1) of HT‐58‐2 cultures with NaBH4 in methanol causes reduction of the peripheral ketone auxochromes, whereupon tolyporphins (predominantly 7,17‐dioxobacteriochlorins) exhibit a bathochromic shift (λabs ˜ 676 → ˜ 700 nm) and chlorophyll a (a 131‐oxochlorin) exhibits a hypsochromic shift (λabs 665 → 634 nm). Fluorescence excitation spectroscopy (at 368 and 491 nm with λem 710 nm) enabled detection of reduced tolyporphins amidst abundant reduced chlorophyll a (1:19 ratio), a detection sensitivity >5 times that without reduction. The resulting assay combines simple sample preparation from non‐axenic cultures at microscale quantities (2 mL, 2 μm), absence of any fractionation procedures, and fluorescence detection. Tolyporphins were readily detected in cultures of HT‐58‐2 at reasonable growth periods in the absence of environmental stressors, which was not possible previously.