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Enzymatic features and biocatalytic applications of modular polyketide synthase domains
Polyketides are a class of secondary metabolites that are notable for their
chemical diversity and therapeutic relevance. They are biosynthesized by polyketide
synthases (PKSs) megasynthase enzymes in an assembly-line fashion. Though the
molecular architectures of polyketides are complex, their biological precursors are
chemically simple. Thus, understanding this powerful biosynthetic machinery is of
interest for synthetic biology and biocatalytic applications. This dissertation presents
three projects that decipher underlying mechanistic features and explore biocatalytic
applications of PKSs.
In modular PKSs, one module corresponds to one round of keto-elongation
followed by modification through the action of Ξ²-carbon processing domains. The first
project employs a system wherein a single module is used in vitro to generate small,
chiral PKS products (triketide lactones). Although triketide lactones are a common output
for PKS enzymology assays, usually they are only observed in trace quantities. In this
study, we performed a number of strategies to scale up the production of triketide
lactones to facilitate their use as chiral building blocks for chemical synthesis. In this
process, we also gained new insights regarding the interacting kinetics and selectivities of
the domains in an in vitro environment.
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The second project focused on the ketoreductase (KR) domain, which sets the
majority of the stereogenic centers within a polyketide, and thus has obvious potential for
biocatalytic applications. This project employs a structure-activity relationship (SAR)-
type approach to dissecting stereocontrol. The SAR results, in concert with
crystallographic data inspired two rational mutations that were sufficient to reverse the
stereoselectivity of a representative KR. Thus, we were able to employ a rational
approach to engineering stereocontrol.
The final project also focuses on the KR domain, however from a subclass of
PKSs termed trans-acyltranferase (AT) PKSs. In contrast to the canonical cis-AT PKSs,
the trans-AT PKSs have more varied modular organizations and architectures. One of
these peculiar organizations one termed a βsplitβ bimodule, wherein domains within a
module are present on different polypeptides. Structural characterization of a KR from a
split bimodule revealed features that may correspond to interpeptide interactions that
afford communication between the two polypeptides of the split bimodule. Additionally,
bioinformatic analysis of KRs from split bimodules reveals a number of diagnostic
sequence motifs.Chemistr
Bacterial vaginosis, vaginal flora patterns and vaginal hygiene practices in patients presenting with vaginal discharge syndrome in The Gambia, West Africa
BACKGROUND: Bacterial vaginosis (BV) β a syndrome characterised by a shift in vaginal flora β appears to be particularly common in sub-Saharan Africa, but little is known of the pattern of vaginal flora associated with BV in Africa. We conducted a study aimed at determining the prevalence of BV and patterns of BV-associated vaginal micro-flora among women with vaginal discharge syndrome (VDS) in The Gambia, West Africa. METHODS: We enrolled 227 women with VDS from a large genito-urinary medicine clinic in Fajara, The Gambia. BV was diagnosed by the Nugent's score and Amsel's clinical criteria. Vaginal swabs were collected for T vaginalis and vaginal flora microscopy, and for Lactobacillus spp, aerobic organisms, Candida spp and BV-associated bacteria (Gardnerella vaginalis, anaerobic bacteria, and Mycoplasma spp) cultures; and cervical swabs were collected for N gonorrhoeae culture and C trachomatis PCR. Sera were tested for HIV-1 and HIV-2 antibodies. Sexual health history including details on sexual hygiene were obtained by standardised questionnaire. RESULTS: BV prevalence was 47.6% by Nugent's score and 30.8% by Amsel's clinical criteria. Lactobacillus spp were isolated in 37.8% of women, and 70% of the isolates were hydrogen-peroxide (H(2)0(2))-producing strains. Prevalence of BV-associated bacteria were: G vaginalis 44.4%; Bacteroides 16.7%; Prevotella 15.2%; Peptostretococcus 1.5%; Mobiluncus 0%; other anaerobes 3.1%; and Mycoplasma hominis 21.4%. BV was positively associated with isolation of G vaginalis (odds-ratio [OR] 19.42, 95%CI 7.91 β 47.6) and anaerobes (P = 0.001 [OR] could not be calculated), but not with M hominis. BV was negatively associated with presence of Lactobacillus (OR 0.07, 95%CI 0.03 β 0.15), and H(2)O(2)-producing lactobacilli (OR 0.12, 95% CI 0.05 β 0.28). Presence of H(2)O(2)-producing lactobacilli was associated with significantly lower prevalence of G vaginalis, anaerobes and C trachomatis. HIV prevalence was 12.8%. Overall, there was no association between BV and HIV, and among micro-organisms associated with BV, only Bacteroides spp. and Prevotella spp. were associated with HIV. BV or vaginal flora patterns were not associated with any of the factors relating to sexual hygiene practices (vaginal douching, menstrual hygiene, female genital cutting). CONCLUSION: In this population, BV prevalence was higher than in corresponding populations in industrialised countries, but the pattern of vaginal micro-flora associated with BV was similar. BV or vaginal flora patterns were not associated with HIV nor with any of the vaginal hygiene characteristics