A Promiscuous Bacterial P450: The Unparalleled Diversity of BM3 in Pharmaceutical Metabolism

From MDPI via Jisc Publications RouterHistory: accepted 2021-10-12, pub-electronic 2021-10-21Publication status: PublishedFunder: Biotechnology and Biological Sciences Research Council; Grant(s): BB/M011208/1CYP102A1 (BM3) is a catalytically self-sufficient flavocytochrome fusion protein isolated from Bacillus megaterium, which displays similar metabolic capabilities to many drug-metabolizing human P450 isoforms. BM3′s high catalytic efficiency, ease of production and malleable active site makes the enzyme a desirable tool in the production of small molecule metabolites, especially for compounds that exhibit drug-like chemical properties. The engineering of select key residues within the BM3 active site vastly expands the catalytic repertoire, generating variants which can perform a range of modifications. This provides an attractive alternative route to the production of valuable compounds that are often laborious to synthesize via traditional organic means. Extensive studies have been conducted with the aim of engineering BM3 to expand metabolite production towards a comprehensive range of drug-like compounds, with many key examples found both in the literature and in the wider industrial bioproduction setting of desirable oxy-metabolite production by both wild-type BM3 and related variants. This review covers the past and current research on the engineering of BM3 to produce drug metabolites and highlights its crucial role in the future of biosynthetic pharmaceutical production

Analysis of meiotic recombination in 22q11.2, a region that frequently undergoes deletions and duplications

BACKGROUND: The 22q11.2 deletion syndrome is the most frequent genomic disorder with an estimated frequency of 1/4000 live births. The majority of patients (90%) have the same deletion of 3 Mb (Typically Deleted Region, TDR) that results from aberrant recombination at meiosis between region specific low-copy repeats (LCRs). METHODS: As a first step towards the characterization of recombination rates and breakpoints within the 22q11.2 region we have constructed a high resolution recombination breakpoint map based on pedigree analysis and a population-based historical recombination map based on LD analysis. RESULTS: Our pedigree map allows the location of recombination breakpoints with a high resolution (potential recombination hotspots), and this approach has led to the identification of 5 breakpoint segments of 50 kb or less (8.6 kb the smallest), that coincide with historical hotspots. It has been suggested that aberrant recombination leading to deletion (and duplication) is caused by low rates of Allelic Homologous Recombination (AHR) within the affected region. However, recombination rate estimates for 22q11.2 region show that neither average recombination rates in the 22q11.2 region or within LCR22-2 (the LCR implicated in most deletions and duplications), are significantly below chromosome 22 averages. Furthermore, LCR22-2, the repeat most frequently implicated in rearrangements, is also the LCR22 with the highest levels of AHR. In addition, we find recombination events in the 22q11.2 region to cluster within families. Within this context, the same chromosome recombines twice in one family; first by AHR and in the next generation by NAHR resulting in an individual affected with the del22q11.2 syndrome. CONCLUSION: We show in the context of a first high resolution pedigree map of the 22q11.2 region that NAHR within LCR22 leading to duplications and deletions cannot be explained exclusively under a hypothesis of low AHR rates. In addition, we find that AHR recombination events cluster within families. If normal and aberrant recombination are mechanistically related, the fact that LCR22s undergo frequent AHR and that we find familial differences in recombination rates within the 22q11.2 region would have obvious health-related implications

Ancestral Origin of the ATTCT Repeat Expansion in Spinocerebellar Ataxia Type 10 (SCA10)

Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant neurodegenerative disease characterized by cerebellar ataxia and seizures. The disease is caused by a large ATTCT repeat expansion in the ATXN10 gene. The first families reported with SCA10 were of Mexican origin, but the disease was soon after described in Brazilian families of mixed Portuguese and Amerindian ancestry. The origin of the SCA10 expansion and a possible founder effect that would account for its geographical distribution have been the source of speculation over the last years. To unravel the mutational origin and spread of the SCA10 expansion, we performed an extensive haplotype study, using closely linked STR markers and intragenic SNPs, in families from Brazil and Mexico. Our results showed (1) a shared disease haplotype for all Brazilian and one of the Mexican families, and (2) closely-related haplotypes for the additional SCA10 Mexican families; (3) little or null genetic distance in small normal alleles of different repeat sizes, from the same SNP lineage, indicating that they are being originated by a single step mechanism; and (4) a shared haplotype for pure and interrupted expanded alleles, pointing to a gene conversion model for its generation. In conclusion, we show evidence for an ancestral common origin for SCA10 in Latin America, which might have arisen in an ancestral Amerindian population and later have been spread into the mixed populations of Mexico and Brazil

Remdesivir-ivermectin combination displays synergistic interaction with improved in vitro activity against SARS-CoV-2

A key element for the prevention and management of COVID-19 is the development of effective therapeutics. Drug combination strategies of repurposed drugs offer several advantages over monotherapies, including the potential to achieve greater efficacy, the potential to increase the therapeutic index of drugs and the potential to reduce the emergence of drug resistance. Here, we report on the in vitro synergistic interaction between two FDA approved drugs, remdesivir and ivermectin resulting in enhanced antiviral activity against SARS-CoV-2. Whilst the in vitro synergistic activity reported here does not support the clinical application of this combination treatment strategy, due to insufficient exposure of ivermectin in vivo, the data do warrant further investigation. Efforts to define the mechanisms underpinning the observed synergistic action, could lead to the development of novel therapeutic treatment strategies

Multiplatform Analysis of 12 Cancer Types Reveals Molecular Classification within and across Tissues of Origin

Recent genomic analyses of pathologically-defined tumor types identify “within-a-tissue” disease subtypes. However, the extent to which genomic signatures are shared across tissues is still unclear. We performed an integrative analysis using five genome-wide platforms and one proteomic platform on 3,527 specimens from 12 cancer types, revealing a unified classification into 11 major subtypes. Five subtypes were nearly identical to their tissue-of-origin counterparts, but several distinct cancer types were found to converge into common subtypes. Lung squamous, head & neck, and a subset of bladder cancers coalesced into one subtype typified by TP53 alterations, TP63 amplifications, and high expression of immune and proliferation pathway genes. Of note, bladder cancers split into three pan-cancer subtypes. The multi-platform classification, while correlated with tissue-of-origin, provides independent information for predicting clinical outcomes. All datasets are available for data-mining from a unified resource to support further biological discoveries and insights into novel therapeutic strategies

Redox driven B12-ligand switch drives CarH photoresponse

Abstract CarH is a coenzyme B12-dependent photoreceptor involved in regulating carotenoid biosynthesis. How light-triggered cleavage of the B12 Co-C bond culminates in CarH tetramer dissociation to initiate transcription remains unclear. Here, a series of crystal structures of the CarH B12-binding domain after illumination suggest formation of unforeseen intermediate states prior to tetramer dissociation. Unexpectedly, in the absence of oxygen, Co-C bond cleavage is followed by reorientation of the corrin ring and a switch from a lower to upper histidine-Co ligation, corresponding to a pentacoordinate state. Under aerobic conditions, rapid flash-cooling of crystals prior to deterioration upon illumination confirm a similar B12-ligand switch occurs. Removal of the upper His-ligating residue prevents monomer formation upon illumination. Combined with detailed solution spectroscopy and computational studies, these data demonstrate the CarH photoresponse integrates B12 photo- and redox-chemistry to drive large-scale conformational changes through stepwise Co-ligation changes