Synthesis of 5-Hydroxyectoine from Ectoine: Crystal Structure of the Non-Heme Iron(II) and 2-Oxoglutarate-Dependent Dioxygenase EctD

As a response to high osmolality, many microorganisms synthesize various types of compatible solutes. These organic osmolytes aid in offsetting the detrimental effects of low water activity on cell physiology. One of these compatible solutes is ectoine. A sub-group of the ectoine producer's enzymatically convert this tetrahydropyrimidine into a hydroxylated derivative, 5-hydroxyectoine. This compound also functions as an effective osmostress protectant and compatible solute but it possesses properties that differ in several aspects from those of ectoine. The enzyme responsible for ectoine hydroxylation (EctD) is a member of the non-heme iron(II)-containing and 2-oxoglutarate-dependent dioxygenases (EC 1.14.11). These enzymes couple the decarboxylation of 2-oxoglutarate with the formation of a high-energy ferryl-oxo intermediate to catalyze the oxidation of the bound organic substrate. We report here the crystal structure of the ectoine hydroxylase EctD from the moderate halophile Virgibacillus salexigens in complex with Fe3+ at a resolution of 1.85 Å. Like other non-heme iron(II) and 2-oxoglutarate dependent dioxygenases, the core of the EctD structure consists of a double-stranded β-helix forming the main portion of the active-site of the enzyme. The positioning of the iron ligand in the active-site of EctD is mediated by an evolutionarily conserved 2-His-1-carboxylate iron-binding motif. The side chains of the three residues forming this iron-binding site protrude into a deep cavity in the EctD structure that also harbours the 2-oxoglutarate co-substrate-binding site. Database searches revealed a widespread occurrence of EctD-type proteins in members of the Bacteria but only in a single representative of the Archaea, the marine crenarchaeon Nitrosopumilus maritimus. The EctD crystal structure reported here can serve as a template to guide further biochemical and structural studies of this biotechnologically interesting enzyme family

Additional file 1: Table S1. of Fatty acid kinase A is an important determinant of biofilm formation in Staphylococcus aureus USA300

Complete list of SNPs in genes representing divergent alleles in UAS391 and TCH1516 and their putative gene functions. TCH1516 was used as a reference for comparison with the allelic change in UAS391. Nucleotide changes in bold red represent the common SNPs in both FPR3757 and TCH1516 as compared to UAS391 (also in Table 2). (XLSX 14 kb

Additional file 4: Figure S2. of Fatty acid kinase A is an important determinant of biofilm formation in Staphylococcus aureus USA300

Multiple alignment of fakA gene sequences in Staphylococcus aureus and Staphylococcus epidermidis. Unique nucleotide variation in SAUSA300_1119 of UAS391 encoding a fatty acid kinase fakA at position 1223940 nt, corresponding to nt 393 of the gene. The respective SAUSA300_1119 allele in UAS391 contains a non-synonymous SNP (Glu to Gly) as compared to strains TCH1516 and FPR3757. The Genbank accession numbers were indicated among these 50 strains. The first 45 strains represent S. aureus, the last 5 strains represent S. epidermidis. (PDF 897 kb

Additional file 3: Figure S1. of Fatty acid kinase A is an important determinant of biofilm formation in Staphylococcus aureus USA300

Comparative genome alignment of USA300, TCH1516, FPR3757 and UAS391 shows high conservation between these genomes. Pink regions show homology and the vertical lines shows conserved blocks. The UAS391 ~13 kb genomic region (from 680369 bp to 693620 bp) is translocated to (1630711 to 1642611bp) in FPR3757. (PDF 116 kb

Additional file 2: Table S2. of Fatty acid kinase A is an important determinant of biofilm formation in Staphylococcus aureus USA300

Complete list of SNPs in genes representing divergent alleles in UAS391 and FPR3757 and their putative gene functions. FPR3757 was used as a reference for comparison with the allelic change in UAS391. Nucleotide changes in bold red represent the common SNPs in both FPR3757 and TCH1516 as compared to UAS391 (also in Table 2). (XLSX 14 kb

Single-molecule sequencing reveals the molecular basis of multidrug-resistance in ST772 methicillin-resistant Staphylococcus aureus

YCT is an Australian National Health and Medical Research Council Career Development Fellow (1065736). DAR was supported in part by National Institutes of Health grant GM080602. SRH, PC, MTGH, JP and SDB were supported by Wellcome Trust grant 098051.Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-associated infection, but there is growing awareness of the emergence of multidrug-resistant lineages in community settings around the world. One such lineage is ST772-MRSA-V, which has disseminated globally and is increasingly prevalent in India. Here, we present the complete genome sequence of DAR4145, a strain of the ST772-MRSA-V lineage from India, and investigate its genomic characteristics in regards to antibiotic resistance and virulence factors.Publisher PDFPeer reviewe