Draft Genome Sequence of Acetobacter aceti Strain 1023, a Vinegar Factory Isolate.

The genome sequence of Acetobacter aceti 1023, an acetic acid bacterium adapted to traditional vinegar fermentation, comprises 3.0 Mb (chromosome plus plasmids). A. aceti 1023 is closely related to the cocoa fermenter Acetobacter pasteurianus 386B but possesses many additional insertion sequence elements.

Transcellular biosynthesis of cysteinyl leukotrienes in vivo during mouse peritoneal inflammation.

Leukotrienes (LTs) are lipid mediators of inflammation formed by enzymatic oxidation of arachidonic acid. One intriguing aspect of LT production is transcellular biosynthesis: cells expressing 5-lipoxygenase (5LO) form LTA(4) and transfer it to cells expressing LTA(4) hydrolase (LTA(4)H) or LTC(4) synthase (LTC(4)S) to produce LTB(4) or LTC(4). This process has been demonstrated in vivo for LTB(4), but not for cysteinyl LTs (cysLTs). We examined transcellular cysLT synthesis during zymosan-induced peritonitis, using bone marrow transplants with transgenic mice deficient in key enzymes of LT synthesis and analyzing all eicosanoids by liquid chromatography/tandem mass spectrometry. WT mice time-dependently produced LTB(4) and cysLTs (LTC(4), LTD(4), and LTE(4)). 5LO(-/-) mice were incapable of producing LTs. WT bone marrow cells restored this biosynthetic ability, but 5LO(-/-) bone marrow did not rescue LT synthesis in irradiated WT mice, demonstrating that bone marrow-derived cells are the ultimate source of all LTs in this model. Total levels of 5LO-derived products were comparable in LTA(4)H(-/-) and WT mice, but were reduced in LTC(4)S(-/-) animals. No differences in prostaglandin production were observed between these transgenic or chimeric mice. Bone marrow cells from LTA(4)H(-/-) or LTC(4)S(-/-) mice injected into 5LO(-/-) mice restored the ability to synthesize LTB(4) and cysLTs, providing unequivocal evidence of efficient transcellular biosynthesis of cysLTs. These results highlight the potential relevance of transcellular exchange of LTA(4) for the synthesis of LTs mediating biological activities during inflammatory events in vivo.

Multiple active site histidine protonation states in Acetobacter aceti N5-carboxyaminoimidazole ribonucleotide mutase detected by REDOR NMR.

Class I PurE (N5-carboxyaminoimidazole mutase) catalyzes a chemically unique mutase reaction. A working mechanistic hypothesis involves a histidine (His45 in Escherichia coli PurE) functioning as a general acid, but no evidence for multiple protonation states has been obtained. Solution NMR is a peerless tool for this task but has had limited application to enzymes, most of which are larger than its effective molecular size limit. Solid-state NMR is not subject to this limit. REDOR NMR studies of a 151 kDa complex of uniformly 15N-labeled Acetobacter aceti PurE (AaPurE) and the active site ligand [6-13C]citrate probed a single ionization equilibrium associated with the key histidine (AaPurE His59). In the AaPurE complex, the citrate central carboxylate C6 13C peak moves upfield, indicating diminution of negative charge, and broadens, indicating heterogeneity. Histidine 15N chemical shifts indicate His59 exists in approximately equimolar amounts of an Ndelta-unprotonated (pyridine-like) form and an Ndelta-protonated (pyrrole-like) form, each of which is approximately 4 A from citrate C6. The spectroscopic data are consistent with proton transfers involving His59 Ndelta that are invoked in the class I PurE mechanism.

Structure of a NADH-insensitive hexameric citrate synthase that resists acid inactivation.

Acetobacter aceti converts ethanol to acetic acid, and strains highly resistant to both are used to make vinegar. A. aceti survives acetic acid exposure by tolerating cytoplasmic acidification, which implies an unusual adaptation of cytoplasmic components to acidic conditions. A. aceti citrate synthase (AaCS), a hexameric type II citrate synthase, is required for acetic acid resistance and, therefore, would be expected to function at low pH. Recombinant AaCS has intrinsic acid stability that may be a consequence of strong selective pressure to function at low pH, and unexpectedly high thermal stability for a protein that has evolved to function at approximately 30 degrees C. The crystal structure of AaCS, complexed with oxaloacetate (OAA) and the inhibitor carboxymethyldethia-coenzyme A (CMX), was determined to 1.85 A resolution using protein purified by a tandem affinity purification procedure. This is the first crystal structure of a "closed" type II CS, and its active site residues interact with OAA and CMX in the same manner observed in the corresponding type I chicken CS.OAA.CMX complex. While AaCS is not regulated by NADH, it retains many of the residues used by Escherichia coli CS (EcCS) for NADH binding. The surface of AaCS is abundantly decorated with basic side chains and has many fewer uncompensated acidic charges than EcCS; this constellation of charged residues is stable in varied pH environments and may be advantageous in the A. aceti cytoplasm.

Biochemical and structural studies of N5-carboxyaminoimidazole ribonucleotide mutase from the acidophilic bacterium Acetobacter aceti.

N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) mutase (PurE) catalyzes the reversible interconversion of acid-labile compounds N5-CAIR and 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). We have examined PurE from the acidophilic bacterium Acetobacter aceti (AaPurE), focusing on its adaptation to acid pH and the roles of conserved residues His59 and His89. Both AaPurE and Escherichia coli PurE showed quasi-reversible acid-mediated inactivation, but wt AaPurE was much more stable at pH 3.5, with a > or = 20 degrees C higher thermal unfolding temperature at all pHs. His89 is not essential and does not function as part of a proton relay system. The kcat pH-rate profile was consistent with the assignment of pK1 to unproductive protonation of bound nucleotide and pK2 to deprotonation of His59. A 1.85 A resolution crystal structure of the inactive mutant H59N-AaPurE soaked in CAIR showed that protonation of CAIR C4 can occur in the absence of His59. The resulting species, modeled as isoCAIR [4(R)-carboxy-5-iminoimidazoline ribonucleotide], is strongly stabilized by extensive interactions with the enzyme and a water molecule. The carboxylate moiety is positioned in a small pocket proposed to facilitate nucleotide decarboxylation in the forward direction (N5-CAIR --> CAIR) [Meyer, E., Kappock, T. J., Osuji, C., and Stubbe, J. (1999) Biochemistry 38, 3012-3018]. Comparisons with model studies suggest that in the reverse (nonbiosynthetic) direction PurE favors protonation of CAIR C4. We suggest that the essential role of protonated His59 is to lower the barrier to decarboxylation by stabilizing a CO2-azaenolate intermediate.