Vital roles of an interhelical insertion in catalase-peroxidase bifunctionality

The loop connecting the F and G helices of catalase-peroxidases contains a approximately 35 amino acid structure (the FG insertion) that is absent from monofunctional peroxidases. These two groups of enzymes share highly similar active sites, yet the monofunctional peroxidases lack appreciable catalase activity. Thus, the FG insertion may serve a role in catalase-peroxidase bifunctionality, despite its peripheral location relative to the active site. We produced a variant of Escherichia coli catalase-peroxidase (KatG) lacking its FG insertion (KatG(DeltaFG)). Absorption spectra indicated the heme environment of KatG(DeltaFG) was highly similar to wild-type KatG, but the variant retained only 0.2% catalase activity. In contrast, the deletion reduced peroxidase activity by only 50%. Kinetic parameters for the peroxidase and residual catalase activities of KatG(DeltaFG) as well as pH dependence studies suggested that the FG insertion supports hydrogen-bonded networks critical for reactions involving H2O2. The structure also appears to regulate access of electron donors to the active site.     

Evidence for distinct mRNAs for ferritin subunits

Poly A enriched RNA from iron loaded HeLa cells and rat liver were translated separately and together in wheat germ lysates to investigate the origins of the H and L subunits of ferritin. Most of the ferritin translated from the HeLa RNA was of the H type, while that from the liver RNA was mostly L type. Mixtures of these RNAs gave $\text{H}\text{L}$ ratios which correlated with the relative amounts of added HeLa and rat RNAs. These results indicate that the H and L subunits of ferritin are not derived by post-translational modification but from distinct mRNA species.     

Diverse and conserved roles of CLE peptides

The function of plant CLAVATA3 (CLV3)/ENDOSPERM SURROUNDING REGION (ESR) (CLE) peptides in shoot meristem differentiation has been expanded in recent years to implicate roles in root growth and vascular development among different CLE family members. Recent evidence suggests that nematode pathogens within plant roots secrete ligand mimics of plant CLE peptides to modify selected host cells into multinucleate feeding sites. This discovery demonstrated an unprecedented adaptation of an animal gene product to functionally mimic a plant peptide involved in cellular signaling for parasitic benefit. This review highlights the diverse and conserved role of CLE peptides in these different contexts.     

Identification and analysis of unitary pseudogenes: historic and contemporary gene losses in humans and other primates

Background  

Unitary pseudogenes are a class of unprocessed pseudogenes without functioning counterparts in the genome. They constitute only a small fraction of annotated pseudogenes in the human genome. However, as they represent distinct functional losses over time, they shed light on the unique features of humans in primate evolution.     

Processed pseudogenes for rat cytochrome c are preferentially derived from one of three alternate mRNAs.

Three cytochrome c mRNAs (1,400, 1,100 and 700 nucleotides) are colinear with RC4, a gene that has introns and correctly encodes cytochrome c. A comparison of RC4 to six nonallelic clones isolated from the rat cytochrome c multigene family demonstrates that all three mRNAs are represented in the genome as processed pseudogenes. Four of the six pseudogenes are derived from the 1,100-nucleotide mRNA, and genomic hybridizations further establish that nearly all of the 30 or so gene family members are also genomic copies of this mRNA despite the equimolar ratio of the three messages in rat tissues. Thus, the surprising multiplicity of cytochrome c sequences in the rat genome is mainly accounted for by the selective use of the 1,100-nucleotide mRNA for the formation of processed pseudogenes. In contrast to 700- and 1,400-nucleotide species which are polyadenylated downstream from AAGUAAA and AAUUAAA, respectively, the 1,100-nucleotide mRNA uses the ubiquitous AAUAAA and also displays a unique stem and loop structure (delta G = -59.4 kJ) centered 37 base pairs upstream from this sequence.     

Evidence for frequent incest in a cooperatively breeding mammal

As breeding between relatives often results in inbreeding depression, inbreeding avoidance is widespread in the animal kingdom. However, inbreeding avoidance may entail fitness costs. For example, dispersal away from relatives may reduce survival. How these conflicting selection pressures are resolved is challenging to investigate, but theoretical models predict that inbreeding should occur frequently in some systems. Despite this, few studies have found evidence of regular incest in mammals, even in social species where relatives are spatio-temporally clustered and opportunities for inbreeding frequently arise. We used genetic parentage assignments together with relatedness data to quantify inbreeding rates in a wild population of banded mongooses, a cooperatively breeding carnivore. We show that females regularly conceive to close relatives, including fathers and brothers. We suggest that the costs of inbreeding avoidance may sometimes outweigh the benefits, even in cooperatively breeding species where strong within-group incest avoidance is considered to be the norm.     

Structural and functional roles of highly conserved serines in human lipoprotein lipase. Evidence that serine 132 is essential for enzyme catalysis

The structure of human lipoprotein lipase was recently deduced from its cDNA sequence. It contains 8 serine residues (residues 45, 132, 143, 172, 193, 244, 251, and 363) that are absolutely conserved in both lipoprotein lipase and hepatic lipase across all species studied. The high homology between lipoprotein lipase, hepatic lipase, and pancreatic lipase suggests that the catalytic functions of these enzymes share a common mechanism and that one of the 8 conserved serines in human lipoprotein lipase must play a catalytic role as does serine 152 in the case of pancreatic lipase (Winkler, F. K., D'Arcy, A., and Hunziker, W. Nature 343, 771-774). We expressed wild-type and site-specific mutants of human lipoprotein lipase in COS cells in vitro. We produced two to four substitution mutants involving each of the 8 serines and assayed a total of 22 mutants for both enzyme activity and the amount of immunoreactive enzyme mass produced. Immunoreactive lipase was detected in all cases. With the exception of Ser132, for each of the 8 serine mutants we studied, at least one of several mutants at each position showed detectable enzyme activity. All three substitution mutants at Ser132, Ser----Thr, Ser----Ala, and Ser----Asp, were totally inactive. Ser132 occurs in the consensus sequence Gly-Xaa-Ser-Xaa-Gly present in all serine proteinases and in human pancreatic lipase. The x-ray crystallography structure of human pancreatic lipase suggests that the analogous serine residue in human pancreatic lipase, Ser152, is the nucleophilic residue essential for catalysis. Our biochemical data strongly support the conclusion that Ser132 in human lipoprotein lipase is the crucial residue required for enzyme catalysis. The observed specific activities of the variants involving the other seven highly conserved serines in human lipoprotein lipase are consistent with the interpretation that this enzyme has a three-dimensional structure very similar to that of human pancreatic lipase.     

Mutagenesis of histidinol dehydrogenase reveals roles for conserved histidine residues.

The dimeric zinc metalloenzyme L-histidinol dehydrogenase (HDH) catalyzes an unusual four-electron oxidation of the amino alcohol histidinol via the histidinaldehyde intermediate to the acid product histidine with the reduction of two molecules of NAD. An essential base, with pKa about 8, is involved in catalysis. Here we report site-directed mutagenesis studies to replace each of the five histidine residues (His-98, His-261, His-326, His-366, and His-418) in Salmonella typhimurium with either asparagine or glutamine. In all cases, the overexpressed enzymes were readily purified and behaved as dimers. Substitution of His-261 and His-326 by asparagine caused about 7000- and 500-fold decreases in kcat, respectively, with little change in KM values. Similar loss of activity was also reported for a H261N mutant Brassica HDH [Nagai, A., and Ohta, D. (1994) J. Biochem. 115, 22-25]. Kinetic isotope effects, pH profiles, substrate rescue, and stopped-flow experiments suggested that His-261 and His-326 are involved in proton transfers during catalysis. Sensitivity to metal ion chelator and decreased affinities for metal ions with substitutions at His-261 and His-418 suggested that these two residues are candidates for zinc ion ligands.