Diversity of microbial community and its metabolic potential for nitrogen and sulfur cycling in sediments of Phu Quoc island, Gulf of Thailand.

Although Phu Quoc island, Gulf of Thailand possesses diverse marine and coastal ecosystems, biodiversity and metabolic capability of microbial communities remain poorly investigated. The aim of our study was to evaluate the biodiversity and metabolic potential of sediment microbial communities in Phu Quoc island. The marine sediments were collected from three different areas and analyzed by using 16S rRNA gene-based amplicon approach. A total of 1,143,939 reads were clustered at a 97% sequence similarity into 8,331 unique operational taxonomic units, representing 52 phyla. Bacteria and archaea occupied averagely around 86% and 14%, respectively, of the total prokaryotic community. Proteobacteria, Planctomycetes, Chloroflexi, and Thaumarchaeota were the dominant phyla in all sediments, which were involved in nitrogen and sulfur metabolism. Sediments harboring of higher nitrogen sources were found to coincide with increased abundance of archaeal phylum Thaumarchaeota. Predictive functional analysis showed high abundance prokaryotic genes associated with nitrogen cycling including nifA-Z, amoABC, nirA, narBIJ, napA, nxrAB, nrfA-K, nirBD, nirS, nirK, norB-Z, nlnA, ald, and ureA-J, based on taxonomic groups detected by 16S rRNA sequencing. Although the key genes involved in sulfur cycling were found to be at low to undetectable levels, the other genes encoding for sulfur-related biological processes were present, suggesting that alternative pathways may be involved in sulfur cycling at our study site. In conclusion, our study for the first time shed light on diversity of microbial communities in Phu Quoc island.

Characterization of a thermophilic cytochrome P450 of the CYP203A subfamily from Binh Chau hot spring in Vietnam.

Cytochromes P450 (CYPs or P450s) comprise a superfamily of heme-containing monooxygenases that are involved in a variety of biological processes. CYPs have broad utilities in industry, but most exhibit low thermostability, limiting their use on an industrial scale. Highly thermostable enzymes can be obtained from thermophiles in geothermal areas, including hot springs, offshore oil-producing wells and volcanoes. Here, we report the identification of a gene encoding for a thermophilic CYP from the Binh Chau hot spring metagenomic database, which was designated as P450-T2. The deduced amino acid sequence showed the highest identity of 73.15% with CYP203A1 of Rhodopseudomonas palustris, supporting that P450-T2 is a member of the CYP203A subfamily. Recombinant protein expression yielded 541 nm. The optimal temperature and pH of P450-T2 were 50 °C and 8.0, respectively. The half-life of P450-T2 was 50.2 min at 50 °C, and its melting temperature was 56.80 ± 0.08 °C. It was found to accept electrons from all tested redox partners systems, with BmCPR-Fdx2 being the most effective partner. Screening for putative substrates revealed binding of phenolic compounds, such as l-mimosine and emodin, suggesting a potential application of this new thermophilic P450 in the production of the corresponding hydroxylated products.

Engineering of CYP106A2 for steroid 9α- and 6ß-hydroxylation.

CYP 106A2 from Bacillus megaterium ATCC 13368 has been described as a 15ß-hydroxylase showing also minor 11α-, 9α- and 6ß-hydroxylase activity for progesterone conversion. Previously, mutant proteins with a changed selectivity towards 11α-OH-progesterone have already been produced. The challenge of this work was to create mutant proteins with a higher regioselectivity towards hydroxylation at positions 9 and 6 of the steroid molecule. 9α-hydroxyprogesterone exhibits pharmaceutical importance, because it is a useful intermediate in the production of physiologically active substances which possess progestational activity. Sixteen mutant proteins were selected from a library containing mutated proteins created by a combination of site-directed and saturation mutagenesis of active site residues. Four mutant proteins out of these catalyzed the conversion of progesterone to 9α-OH-progesterone as a main product. For further optimization site-directed mutagenesis was performed. The introduction of seven mutations (D217V, A243V, A106T, F165L, T89N, T247V or T247W) into these four mutant proteins led to 28 new variants, which were also used for an in vivo conversion of progesterone. The best mutant protein, F165L/A395E/G397V, showed a ten-fold increase in the selectivity towards progesterone 9α-hydroxylation compared with the wild type CYP106A2. Also 6ß-OH-progesterone is a pharmaceutically important compound, especially as intermediate for the production of drugs against breast cancer. For the rational design of mutant proteins with 6ß-selectivity, docking of the 3D-structure of CYP106A2 with progesterone was performed. The introduction of three mutations (T247A, A243S, F173A) led to seven new mutant proteins. Clone A243S showed the greatest improvement in 6ß-selectivity being more than ten-fold. Finally, an in vivo conversion of 11-deoxycorticosterone (DOC), testosterone and cortisol with the best five mutant proteins displaying 9α- or 6ß-hydroxylation, respectively, of progesterone was performed to investigate whether the introduced mutations also effected the conversion of other substrates.

Novel homozygous p.Y395X mutation in the CYP11B1 gene found in a Vietnamese patient with 11ß-hydroxylase deficiency.

CONTEXT: The deficiency of steroid 11ß-hydroxylase is caused by mutations in the CYP11B1 gene and is the second major form of congenital adrenal hyperplasia associated with hypertension. OBJECTIVE: The objective of this study was to screen the CYP11B1 gene for mutations in one Vietnamese male suffering from congenital adrenal hyperplasia. PATIENT: The patient (46,XY) had congenital adrenal hyperplasia. The clinical manifestations presented precocious puberty, hyper-pigmentation and high blood pressure at 4 years. RESULTS: The patient was a homozygous carrier of a novel mutation located in exon 7 containing a premature stop codon instead of tyrosine at 395 (p.Y395X). CONCLUSION: We have identified a novel mutant of the CYP11B1 gene in one Vietnamese family associated with phenotypes of congenital adrenal hyperplasia. The mutant gene p.Y395X produces a truncated form of the polypeptide and abolishes the enzyme activities, leading to a severe phenotype of congenital adrenal hyperplasia.

Changing the regioselectivity of a P450 from C15 to C11 hydroxylation of progesterone.

CYP106A2 is known as a 15ß-hydroxylase, but also shows minor 11α-hydroxylase activity for progesterone. 11α-Hydroxyprogesterone is an important pharmaceutical compound with anti-androgenic and blood-pressure-regulating activity. This work therefore focused on directing the regioselectivity of the enzyme towards hydroxylation at position 11 in the C ring of the steroid through a combination of saturation mutagenesis and rational site-directed mutagenesis. With the aid of data from a homology model of CYP106A2 containing docked progesterone, together with site-directed mutagenesis of active-site residues (Lisurek et al. ChemBioChem 2008, 9, 1439-1449), a saturation mutagenesis library at positions A395 and G397 was created. Screening of the library identified the mutants A395I and A395W/G397K as having 11α-hydroxylase activities 8.9 and 11.5 times higher than that of the wild type (WT). In the next step, additional mutations were integrated by a rational site-directed mutagenesis approach to increase the catalytic efficiency. Of the 40 candidates analyzed, the mutants A106T/A395I, A106T/A395I/R409L, and T89N/A395I turned out to display increased 11α-hydroxylase selectivities and activities relative to the WT (14.3-, 12.6-, and 11.8-fold increases in selectivity and 39.3-, 108-, and 24.4- in k(cat)/K(m)). In the last step of the study, the best mutants were applied in a whole-cell biotransformation. In these experiments the production (percentage) of 15ß-hydroxyprogesterone decreased from 50.4 % (wild type) to 4.8 % (mutant T89N/A395I), whereas that of 11α-hydroxyprogesterone increased from 27.7 to 80.9 %, thus demonstrating an impressive regioselectivity.