Bacterial enzymes for dissimilatory sulfate reduction in a marine microbial mat (Black Sea) mediating anaerobic oxidation of methane.

Anaerobic oxidation of methane (AOM) with sulfate is catalysed by microbial consortia of archaea and bacteria affiliating with methanogens and sulfate-reducing Deltaproteobacteria respectively. There is evidence that methane oxidation is catalysed by enzymes related to those in methanogenesis, but the enzymes for sulfate reduction coupled to AOM have not been examined. We collected microbial mats with high AOM activity from a methane seep in the Black Sea. The mats consisted mainly of archaea of the ANME-2 group and bacteria of the Desulfosarcina-Desulfococcus group. Cell-free mat extract contained activities of enzymes involved in sulfate reduction to sulfide: ATP sulfurylase (adenylyl : sulfate transferase; Sat), APS reductase (Apr) and dissimilatory sulfite reductase (Dsr). We partially purified the enzymes by anion-exchange chromatography. The amounts obtained indicated that the enzymes are abundant in the mat, with Sat accounting for 2% of the soluble mat protein. N-terminal amino acid sequences of purified proteins suggested similarities to the corresponding enzymes of known species of sulfate-reducing bacteria. The deduced amino acid sequence of PCR-amplified genes of the Apr subunits is similar to that of Apr of the Desulfosarcina/Desulfococcus group. These results indicate that the major enzymes involved in sulfate reduction in the Back Sea microbial mats are of bacterial origin, most likely originating from the bacterial partner in the consortium.

Purification, crystallization and preliminary X-ray analysis of the dissimilatory sulfite reductase from Desulfovibrio vulgaris Miyazaki F.

Dissimilatory sulfite reductase (Dsr) plays an important role in sulfate respiration in many sulfate-reducing bacteria. Dsr from Desulfovibrio vulgaris Miyazaki F has been purified and crystallized at 277 K using the sitting-drop vapour-diffusion method with PEG 3350 and potassium thiocyanate as precipitants. A data set was collected to 3.7 Šresolution from a single crystal at 100 K using synchrotron radiation. The Dsr crystal belonged to space group P4(1)2(1)2, with unit-cell parameters a = b = 163.26, c = 435.32 Å. The crystal structure of Dsr was determined by the molecular-replacement method based on the three-dimensional structure of Dsr from D. vulgaris Hildenborough. The crystal contained three α(2)ß(2)γ(2) units per asymmetric unit, with a Matthews coefficient (V(M)) of 2.35 Å(3) Da(-1); the solvent content was estimated to be 47.7%.

Purification, crystallization and preliminary crystallographic analysis of a dissimilatory DsrAB sulfite reductase in complex with DsrC.

Dissimilatory sulfite reductase (dSiR, DsrAB) is a key protein in dissimilatory sulfur metabolism, one of the earliest types of energy metabolism to be traced on earth. dSirs are large oligomeric proteins around 200kDa forming an alpha(2)beta(2) arrangement and including a unique siroheme-[4Fe-4S] coupled cofactor. Here, we report the purification, crystallization and preliminary X-ray diffraction analysis of dSir isolated from Desulfovibrio vulgaris Hildenborough, also known as desulfoviridin. In this enzyme the DsrAB protein is associated with DsrC, a protein of unknown function that is believed to play an important role in the sulfite reduction. Crystals belong to the monoclinic space group P2(1) with unit-cell parameters a=122.7, b=119.4 and c=146.7A and beta =110.0 degrees , and diffract X-rays to 2.8A on a synchrotron source.