Normal and Aberrant Methyltransferase Activities Give Insights into the Final Steps of Dynemicin A Biosynthesis.

The naturally occurring enediynes are notable for their complex structures, potent DNA cleaving ability, and emerging usefulness in cancer chemotherapy. They can be classified into three distinct structural families, but all are thought to originate from a common linear C15-heptaene. Dynemicin A (DYN) is the paradigm member of anthraquinone-fused enediynes, one of the three main classes and exceptional among them for derivation of both its enediyne and anthraquinone portions from this same early biosynthetic building block. Evidence is growing about how two structurally dissimilar, but biosynthetically related, intermediates combine in two heterodimerization reactions to create a nitrogen-containing C30-coupled product. We report here deletions of two genes that encode biosynthetic proteins that are annotated as S-adenosylmethionine (SAM)-dependent methyltransferases. While one, DynO6, is indeed the required O-methyltransferase implicated long ago in the first studies of DYN biosynthesis, the other, DynA5, functions in an unanticipated manner in the post-heterodimerization events that complete the biosynthesis of DYN. Despite its removal from the genome of Micromonospora chersina, the ΔdynA5 strain retains the ability to synthesize DYN, albeit in reduced titers, accompanied by two unusual co-metabolites. We link the appearance of these unexpected structures to a substantial and contradictory body of other recent experimental data to advance a biogenetic rationale for the downstream steps that lead to the final formation of DYN. A sequence of product-forming transformations that is in line with new and existing experimental results is proposed and supported by a model reaction that also encompasses the formation of the crucial epoxide essential for the activation of DYN for DNA cleavage.

Gene cluster conservation provides insight into cercosporin biosynthesis and extends production to the genus Colletotrichum.

Species in the genus Cercospora cause economically devastating diseases in sugar beet, maize, rice, soy bean, and other major food crops. Here, we sequenced the genome of the sugar beet pathogen Cercospora beticola and found it encodes 63 putative secondary metabolite gene clusters, including the cercosporin toxin biosynthesis (CTB) cluster. We show that the CTB gene cluster has experienced multiple duplications and horizontal transfers across a spectrum of plant pathogenic fungi, including the wide-host range Colletotrichum genus as well as the rice pathogen Magnaporthe oryzae Although cercosporin biosynthesis has been thought to rely on an eight-gene CTB cluster, our phylogenomic analysis revealed gene collinearity adjacent to the established cluster in all CTB cluster-harboring species. We demonstrate that the CTB cluster is larger than previously recognized and includes cercosporin facilitator protein, previously shown to be involved with cercosporin autoresistance, and four additional genes required for cercosporin biosynthesis, including the final pathway enzymes that install the unusual cercosporin methylenedioxy bridge. Lastly, we demonstrate production of cercosporin by Colletotrichum fioriniae, the first known cercosporin producer within this agriculturally important genus. Thus, our results provide insight into the intricate evolution and biology of a toxin critical to agriculture and broaden the production of cercosporin to another fungal genus containing many plant pathogens of important crops worldwide.

Examining Heterodimerization by Aryl C-N Coupling in Dynemicin Biosynthesis.

Distinct among the enediyne antitumor antibiotics, the dynemicin subgroup is comprised of two discrete halves, an enediyne and an anthraquinone, but each is ultimately derived from the same linear ß-hydroxyhexaene precursor. The linkage of these two halves by an aryl C-N bond is examined here using a variety of experimental approaches. We demonstrate that this heterodimerization is specific for anthracenyl iodide as the corresponding bromo- and amino-substituted anthracenes do not support dynemicin biosynthesis. Furthermore, biochemical experiments and chemical model reactions support an SRN1 mechanism for the aryl C-N coupling in which electron transfer occurs to the iodoanthracene, followed by loss of an anthracenyl iodide and partition of the resulting aryl radical between C-N coupling and reduction by hydrogen abstraction. An enzyme pull-down experiment aiming to capture the protein(s) involved in the coupling reaction is described in which two proteins, Orf14 and Orf16, encoded by the dynemicin biosynthetic gene cluster, are specifically isolated. Deletion of orf14 from the genome abolished dynemicin production accompanied by a 3-fold increased accumulation of the iodoanthracene coupling partner, indicating the plausible involvement of this protein in the heterodimerization process. On the other hand, the deletion of orf16 only reduced dynemicin production to 55%, implying a noncatalytic, auxiliary role of the protein. Structural comparisons using AlphaFold imply key similarities between Orf14 and X-ray crystal structures of several proteins from enediyne BGCs believed to bind hydrophobic polyene or enediyne motifs suggest Orf14 templates aryl C-N bond formation during the central heterodimerization in dynemicin biosynthesis.

Dynemicin A Derivatives as Potential Cancer Chemotherapeutics by Mutasynthesis.

The enediyne antitumor antibiotics have remarkable structures and exhibit potent DNA cleavage properties that have inspired continued interest as cancer therapeutics. Their complex structures and high reactivity, however, pose formidable challenges to their production and development in the clinic. We report here proof-of-concept studies using a mutasynthesis strategy to combine chemical synthesis of select modifications to a key iodoanthracene-γ-thiolactone intermediate in the biosynthesis of dynemicin A and all other known anthraquinone-fused enediynes (AFEs). By chemical complementation of a mutant bacterial producer that is incapable of synthesizing this essential building block, we show that derivatives of dynemicin can be prepared substituted in the A-ring of the anthraquinone motif. In the absence of competition from native production of this intermediate, the most efficient utilization of these externally-supplied structural analogues for precursor-directed biosynthesis becomes possible. To achieve this goal, we describe the required Δorf15 blocked mutant and a general synthetic route to a library of iodoanthracene structural variants. Their successful incorporation opens the door to enhancing DNA binding and tuning the bioreductive activation of the modified enediynes for DNA cleavage.

Finger dermatoglyphic variations in Rengma Nagas of Nagaland India.

The Rengma Nagas are one of the major Mongoloid tribal populations in the North-Eastern state of Nagaland in India. Population variation and sexual dimorphism in respect of finger dermatoglyphic characteristics in 207 adult individuals (104 males and 103 females) are reported in this present context. Frequency distribution of finger pattern types in different digits (both left and right sides combined) showed that whorls were the most prevalent patterns among both males (52.19%) and females (55.69%), followed by loops (47.70% in males and 42.81% in females). Significant sex differences in Dankmeijer Index (t = 1.47; p < 0.0001) and finger wise variations of total finger ridge count (TFRC) and absolute finger ridge count (AFRC) in both the sexes were recorded. However, in cases of the frequencies of finger dermatoglyphic pattern types, Pattern Intensity Index in fingers, TFRC and AFRC no significant sex differences were observed.

Exploring Fungal Polyketide C-Methylation through Combinatorial Domain Swaps.

Polyketide C-methylation occurs during a programmed sequence of dozens of reactions carried out by multidomain polyketide synthases (PKSs). Fungal PKSs perform these reactions iteratively, where a domain may be exposed to and act upon multiple enzyme-tethered intermediates during biosynthesis. We surveyed a collection of C-methyltransferase (CMeT) domains from nonreducing fungal PKSs to gain insight into how different methylation patterns are installed. Our in vitro results show that control of methylation resides primarily with the CMeT, and CMeTs can intercept and methylate intermediates from noncognate nonreducing PKS domains. Furthermore, the methylation pattern is likely imposed by a competition between methylation or ketosynthase-catalyzed extension for each intermediate. Understanding site-specific polyketide C-methylation may facilitate targeted C-C bond formation in engineered biosynthetic pathways.

In vitro study to compare sensitivity of amoxicillin+clavulanic acid and cefpodoxime+clavulanic acid among beta-lactamase positive clinical isolates of gram-positive and gram-negative pathogens.

The present study was carried out to compare the in vitro sensitivity of cefpodoxime + clavulanic acid and amoxicillin + clavulanic acid against 55 Gram-positive and 123 Gram-negative beta-lactamase positive clinical isolates. Micro-organisms isolated from different clinical specimens were tested for beta-lactamase/ESBL by using nitrocefin disc test and for metallo beta-lactamase by using double disc synergy test. A total of 299 (93 Gram-positive and 206 Gram-negative) clinical isolates were tested for beta-lactamase. Among 93 Gram-positive clinical isolates 25 (78.12%) out of 32 coagulase positive S. aureus, 23 (60.52%) out of 38 coagulase negative S aureus, 7 (63.63%) out of 11 enterococci and 0 (0%) out of 12 Strept pneumoniae were positive for beta-lactamase /ESBL. Notably Strept pneumoniae was found to be beta-lactamase/ESBL negative. Among 206 Gram-negative clinical isolates, 25 (69.44%) out of 36 acinetobacter spp, 20 (41.66%) out of 48 Branhamella catarrhalis, 24 (64.86%) out of 37 E. coli, 7 (46.66%) out of 15 H influenzae and 22 (62.85%) out of 35 proteus were positive for beta-lactamase/ ESBL/metallo beta-lactamase. Positive strains were tested for comparative sensitivity to amoxicillin+ clavulanic acid and cefpodoxime+clavulanic acid by Kirby Bauer disc diffusion method. As regards comparative sensitivity among beta-lactamase/ESBL positive Gram-positive strains, 84% and 92% strains of coagulase positive S aureus, 65.21% and 86.95% strains of coagulase negative S. aureus, 83.33% and 100% strains of Strept pneumoniae and 71.42% and 100% strains of enterococci were found sensitive to amoxicillin +clavulanic acid and cefpodoxime + clavulanic acid respectively. Sensitivity to amoxicillin+ clavulanic acid and cefpodoxime +clavulanic acid among beta lactamase/ESBL positive Gram-negative strains of acinetobacter spp, Branhamella catarrhalis, E. coli, H. influenzae and proteus spp were found to be 20% and 28%, 100% and 100%, 50% and 75%, 71.42% and 100%, 50% and 68.18% respectively. This study demonstrated that cefpodoxime +clavulanic acid combination has more potent in vitro activity in comparison to amoxicillin+ clavulanic acid combination against beta-lactamase producing strains of Gram-positive and Gram-negative bacteria. Given this broad spectrum of activity, cefpodoxime+clavulanic acid appears well suited for use in the treatment of a variety of healthcare-associated infections.

Evaluation of sensitivity of different organisms to cefepime and tazobactum (megapime XP) in comparison to cefepime and ceftazidime.

The present study was carried out to evaluate the sensitivity of different organisms to cefepime and tazobactum (megapime XP) in comparison to cefepime and ceftazidime. Samples were collected from clinical specimens and micro-organisms were isolated from clinical specimens. Strains of micro-organisms isolated from clinical specimens were identified using standard methods like morphology, colony characteristics and biochemical reactions. Sensitivity was carried out using Kirby Bauer disc diffusion method. Minimum inhibitory concentrations (MICs) for all the antibiotics were carried out as per guideline of Clinical and Laboratory Standard Institute (CLSI). The results of the study demonstrated that cefepime and tazobactum exhibits good activity against Gram +ve and Gram-ve organisms.