Cystathionine b-lyase (CBL) catalyzes the hydrolysis of L-cystathionine (L-Cth) to produce L-homocysteine, pyruvate, and ammonia. A series of active-site mutants of Escherichia coli CBL (eCBL) was constructed to investigate the roles of residues R58, R59, D116, W340, and R372 in catalysis and inhibition by aminoethoxyvinylglycine (AVG). The effects of these mutations on the k cat /K LÀCth m for the b-elimination reaction range from a reduction of only 3-fold for D116A and D116N to 6 orders of magnitude for the R372L and R372A mutants. The order of importance of these residues for the hydrolysis of L-Cth is: R372 >> R58 > W340 % R59 > D116. Comparison of the kinetic parameters for L-Cth hydrolysis with those for inhibition of eCBL by AVG demonstrates that residue R58 tethers the distal carboxylate group of the substrate and confirms that residues W340 and R372 interact with the a-carboxylate moiety. The increase in the pK a of the acidic limb and decrease in the pK a of the basic limb of the k cat /K LÀCth m versus pH profiles of the R58K and R58A mutants, respectively, support a role for this residue in modulating the pK a of an active-site residue.

Cystathionine c-synthase (CGS) catalyzes the condensation of O-succinyl-L-homoserine (L-OSHS) and L-cysteine (L-Cys), to produce L-cystathionine (L-Cth) and succinate, in the first step of the bacterial transsulfuration pathway. In the absence of L-Cys, the enzyme catalyzes the futile a,c-elimination of L-OSHS, yielding succinate, a-ketobutyrate, and ammonia. A series of 16 sitedirected variants of Escherichia coli CGS (eCGS) was constructed to probe the roles of active-site residues D45, Y46, R48, R49, Y101, R106, N227, E325, S326, and R361. The effects of these substitutions on the catalytic efficiency of the a,c-elimination reaction range from a reduction of only~2-fold for R49K and the E325A,Q variants to 310-and 760-fold for R361K and R48K, respectively. A similar trend is observed for the k cat /K l-OSHS m of the physiological, a,c-replacement reaction. The results of this study suggest that the arginine residues at positions 48, 106 and 361 of eCGS, conserved in bacterial CGS sequences, tether the distal and a-carboxylate moieties, respectively, of the L-OSHS substrate. In contrast, with the exception of the 13-fold increase observed for R106A, the K l-Cys m is not markedly affected by the site-directed replacement of the residues investigated. The decrease in k cat observed for the S326A variant reflects the role of this residue in tethering the side chain of K198, the catalytic base. Although no structures exist of eCGS bound to active-site ligands, the roles of individual residues is consistent with the structures inhibitor complexes of related enzymes. Substitution of D45, E325, or Y101 enables a minor transamination activity for the substrate L-Ala.

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• Premise of the study: Nuclear microsatellite markers were developed for Lobelia inflata (Campanulaceae), an obligately self-fertilizing plant species, for use in the study of temporal fluctuation in allele frequency and of the genetic structure within and among populations.• Methods and Results: We developed 28 primer pairs for L. inflata, all of which amplify CT dinucleotide repeats. We evaluated amplification of these loci in 53 L. inflata individuals at three sites in eastern North America and found that 24 loci showed microsatellite polymorphism. We also found that 16 loci amplified successfully in L. cardinalis, and 11 amplified successfully in L. siphilitica.• Conclusions: These primers will be useful for assessing allelic diversity within and among populations of L. inflata, and show potential for use in congeneric species.

Increasing evidence suggests that mitochondria play a critical role in driving innate immune responses against bacteria and viruses. However, it is unclear if differential reprogramming of mitochondrial function contributes to the fine tuning of pathogen specific immune responses. Here, we found that TLR3 and TLR4 engagement on murine bone marrow derived macrophages was associated with differential remodeling of electron transport chain complex expression. This remodeling was associated with differential accumulation of mitochondrial and cytosolic ROS, which were required to support ligand specific inflammatory and antiviral cytokine production. We also found that the magnitude of TLR3, but not TLR4, responses were modulated by glucose availability. Under conditions of low glucose, TLR3 engagement was associated with increased ETC complex III expression, increased mitochondrial and cytosolic ROS and increased inflammatory and antiviral cytokine production. This amplification was selectively reversed by targeting superoxide production from the outer Q-binding site of the ETC complex III. These results suggest that ligand specific modulation of the ETC may act as a rheostat that fine tunes innate immune responses via mitochondrial ROS production. Modulation of these processes may represent a novel mechanism to modulate the nature as well as the magnitude of antiviral vs. inflammatory immune responses.