Crystal structure of oleate hydratase from Stenotrophomonas sp. KCTC 12332 reveals conformational plasticity surrounding the FAD binding site.

Unsaturated fatty acids are toxic to various bacteria, causing their death or growth inhibition. To prevent this toxicity, unsaturated fatty acids should be converted into saturated fatty acids via hydrogenation reaction, which is the complete reduction of double bonds on the carbon chain. In a recent report, we observed that Stenotrophomonas sp. KCTC 12332 exhibited a high biotransformation activity of oleic acid (OA) in 10-hydroxystearic acid and identified the gene encoding oleate hydratase (OhySt) by complete genomic analysis. In the present study, to further investigate the structural features of OhySt, the recombinant protein was expressed in Escherichia coli, and then purified and crystallized. Biochemical assay showed that OhySt produces 10-hydroxystearic acid in a flavin adenosine dinucleotide (FAD)-dependent manner, indicating that it requires FAD as a cofactor. The OhySt structure, which is determined in its apo state, allows for a structural comparison with the previously reported FAD bound structure of oleate hydratase. The comparison of structures indicates remarkable conformational change of the loop region surrounding the FAD molecule upon binding of FAD. This change forces one of the important catalytic residues into position for catalysis.

Complete genome sequence of Stenotrophomonas sp. KCTC 12332, a biotechnological potential bacterium.

Hydroxy fatty acids are used in various industries due to their availability, and in particular, Stenotrophomonas sp. has been regarded as a potential candidate for biotechnological applications, including biotransformation that hydrate unsaturated fatty acids into their derivatives. Here we complete the genome sequence of Stenotrophomonas sp. KCTC 12332 which has a size of 4,541,594bp (G+C content of 63.83%) with 3790 coding DNA sequences (CDSs), 67 tRNA and 3 rRNA operons. The genome contains gene encoding oleate hydratase that can convert oleic acid into 10-hydroxyoctadecanoic acid.

Structural insight into the substrate inhibition mechanism of NADP(+)-dependent succinic semialdehyde dehydrogenase from Streptococcus pyogenes.

Succinic semialdehyde dehydrogenases (SSADHs) are ubiquitous enzymes that catalyze the oxidation of succinic semialdehyde (SSA) to succinic acid in the presence of NAD(P)(+), and play an important role in the cellular mechanisms including the detoxification of accumulated SSA or the survival in conditions of limited nutrients. Here, we report the inhibitory properties and two crystal structures of SSADH from Streptococcus pyogenes (SpSSADH) in a binary (ES) complex with SSA as the substrate and a ternary (ESS) complex with the substrate SSA and the inhibitory SSA, at 2.4 Å resolution for both structures. Analysis of the kinetic inhibitory parameters revealed significant substrate inhibition in the presence of NADP(+) at concentrations of SSA higher than 0.02 mM, which exhibited complete uncompetitive substrate inhibition with the inhibition constant (Ki) value of 0.10 ± 0.02 mM. In ES-complex of SpSSADH, the SSA showed a tightly bound bent form nearby the catalytic residues, which may be caused by reduction of the cavity volume for substrate binding, compared with other SSADHs. Moreover, structural comparison of ESS-complex with a binary complex with NADP(+) of SpSSADH indicated that the substrate inhibition was induced by the binding of inhibitory SSA in the cofactor-binding site, instead of NADP(+). Our results provide first structure-based molecular insights into the substrate inhibition mechanism of SpSSADH as the Gram-positive bacterial SSADH.

Kinetic and structural characterization for cofactor preference of succinic semialdehyde dehydrogenase from Streptococcus pyogenes.

The γ-Aminobutyric acid (GABA) that is found in prokaryotic and eukaryotic organisms has been used in various ways as a signaling molecule or a significant component generating metabolic energy under conditions of nutrient limitation or stress, through GABA catabolism. Succinic semialdehyde dehydrogenase (SSADH) catalyzes the oxidation of succinic semialdehyde to succinic acid in the final step of GABA catabolism. Here, we report the catalytic properties and two crystal structures of SSADH from Streptococcus pyogenes (SpSSADH) regarding its cofactor preference. Kinetic analysis showed that SpSSADH prefers NADP(+) over NAD(+) as a hydride acceptor. Moreover, the structures of SpSSADH were determined in an apo-form and in a binary complex with NADP(+) at 1.6 Šand 2.1 Šresolutions, respectively. Both structures of SpSSADH showed dimeric conformation, containing a single cysteine residue in the catalytic loop of each subunit. Further structural analysis and sequence comparison of SpSSADH with other SSADHs revealed that Ser158 and Tyr188 in SpSSADH participate in the stabilization of the 2'-phosphate group of adenine-side ribose in NADP(+). Our results provide structural insights into the cofactor preference of SpSSADH as the gram-positive bacterial SSADH.

The structure of a shellfish specific GST class glutathione S-transferase from antarctic bivalve Laternula elliptica reveals novel active site architecture.

Glutathione-S-transferases have been identified in all the living species examined so far, yet little is known about their function in marine organisms. In a previous report, the recently identified GST from Antarctic bivalve Laternula elliptica (LeGST) was classified into the rho class GST, but there are several unique features of LeGST that may justify reclassification, which could represent specific shellfish GSTs. Here, we determined the crystal structure of LeGST, which is a shellfish specific class of GST. The structural analysis showed that the relatively open and wide hydrophobic H-site of the LeGST allows this GST to accommodate various substrates. These results suggest that the H-site of LeGST may be the result of adaptation to their environments as sedentary organisms.

Crystallization and preliminary X-ray crystallographic studies of succinic semialdehyde dehydrogenase from Streptococcus pyogenes.

Succinic semialdehyde dehydrogenase (SSADH) plays a critical role in the metabolism of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) and catalyzes the NAD(P)(+)-coupled oxidation of succinic semialdehyde (SSA) to succinic acid (SA). SSADH from Streptococcus pyogenes has been purified and crystallized as the apoenzyme and in a complex with NAD(+). The crystals of native and NAD(+)-complexed SSADH diffracted to resolutions of 1.6 and 1.7 Å, respectively, using a synchrotron-radiation source. Both crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 93.3, b = 100.3, c = 105.1 Å for the native crystal and a = 93.3, b = 100.3, c = 105.0 Å for the complex crystal. Preliminary molecular replacement confirmed the presence of one dimer in both crystals, corresponding to a Matthews coefficient (V(M)) of 2.37 Å(3) Da(-1) and a solvent content of 48.0%.

Crystallization and preliminary X-ray crystallographic studies of the rho-class glutathione S-transferase from the Antarctic clam Laternula elliptica.

Glutathione S-transferases are involved in phase II detoxification processes and catalyze the nucleophilic attack of the tripeptide glutathione on a wide range of endobiotic and xenobiotic electrophilic substrates. The rho-class glutathione S-transferase from Laternula elliptica was overexpressed in Escherichia coli, purified and crystallized with two substrates: glutathione and 1-chloro-2,4-dinitrobenzene (CDNB). Diffraction data were collected to 2.20 A resolution for the glutathione-complex crystals and to 2.00 A resolution for the CDNB-complex crystals using a synchrotron-radiation source. Both crystals belonged to the C-centred monoclinic space group C2. The unit-cell parameters for the CDNB-complex crystals were a = 89.66, b = 59.27, c = 55.45 A, beta = 124.52 degrees . The asymmetric unit contained one molecule, with a corresponding V(M) of 2.36 A(3) Da(-1) and a solvent content of 47.8%.

Overexpression, purification, and preliminary X-ray crystallographic analysis of human brain-type creatine kinase.

Creatine kinase (CK; E.C. 2.7.3.2) is an important enzyme that catalyzes the reversible transfer of a phosphoryl group from ATP to creatine in energy homeostasis. The brain-type cytosolic isoform of creatine kinase (BB-CK), which is found mainly in the brain and retina, is a key enzyme in brain energy metabolism, because high-energy phosphates are transferred through the creatine kinase/phosphocreatine shuttle system. The recombinant human BB-CK protein was overexpressed as a soluble form in Escherichia coli and crystallized at 22 degrees C using PEG 4000 as a precipitant. Native X-ray diffraction data were collected to 2.2 A resolution using synchrotron radiation. The crystals belonged to the tetragonal space group P43212, with cell parameters of a=b=97.963, c= 164.312 A, and alpha=beta=gamma=90 degrees. The asymmetric unit contained two molecules of CK, giving a crystal volume per protein mass (Vm) of 1.80 A3 Da-1 and a solvent content of 31.6%.