Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants

Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx mutants to probe this region and to identify the carboxylate residue implicated in catalysis. The pH profile of the D241A CsOxOx mutant suggests that the protonation state of aspartic acid 241 is mechanistically significant and that catalysis takes place at the N-terminal Mn binding site. The observation that the D241S CsOxOx mutation eliminates Mn binding to both the N- and C- terminal Mn binding sites suggests that both sites must be intact for Mn incorporation into either site. The introduction of a proton donor into the N-terminal Mn binding site (CsOxOx A242E mutant) does not affect reaction specificity. Mutation of conserved arginine residues further support that catalysis takes place at the N-terminal Mn binding site and that both sites must be intact for Mn incorporation into either site.     

Autoamplification of a two-component regulatory system results in "learning" behavior

We have tested the hypothesis that the autoamplification of two-component regulatory systems results in "learning" behavior, i.e., that bacteria respond faster or more extensively to a signal when a similar signal has been perceived in the past. Indeed, the induction of alkaline phosphatase activity upon phosphate limitation was faster if the cultures had been limited for phosphate previously, and this faster response correlated with the autoamplification of the cognate two-component system.     

Antigen-antibody immune complexes empower dendritic cells to efficiently prime specific CD8+ CTL responses in vivo

Dendritic cells (DCs) require a maturation signal to acquire efficient CTL-priming capacity. In vitro FcgammaR-mediated internalization of Ag-Ab immune complexes (ICs) can induce maturation of DCs. In this study, we show that IC-induced DC maturation in vitro enables DCs to prime peptide-specific CD8+ CTLs in vivo, independently of CD4+ Th cells. Importantly, OVA/anti-OVA IC-treated DCs not only primed CD8+ CTLs to an exogenously loaded peptide nonrelated to OVA, but also efficiently primed CTLs against the dominant CTL epitope derived from the OVA Ag present in the ICs. Our studies show that ICs fulfill a dual role in priming of CD8+ CTL responses to exogenous Ags: enhancement of Ag uptake by DCs and activation of DCs, resulting in "license to kill." These findings indicate that the presence of specific Abs can crucially affect the induction of cytotoxic cellular responses.     

Effect of protein restriction on sulfur amino acid catabolism in insulin-dependent diabetes mellitus

Persons with conventionally treated insulin-dependent diabetes mellitus (IDDM) appear to be impaired in their ability to reduce fed-state urea production appropriately in response to dietary protein restriction (Hoffer LJ, Taveroff A, and Schiffrin A. Am J Physiol 272: E59-E67, 1997). To determine whether these conclusions apply to whole body sulfur amino acid (SAA) catabolism, we used samples from this protocol to measure daily urinary sulfate excretion and fed-state sulfate production after a high-protein test meal before and after dietary protein restriction. Eight normal subjects and six IDDM subjects treated with twice-daily intermediate- and short-acting insulin consumed a mixed test meal containing 0.50 g protein/kg after adaptation to 4 days of high protein intake (1.28 g protein/kg body wt) and again after 5 days of dietary protein restriction (0.044 g/kg). Adaptation to protein restriction decreased daily urinary sulfate and urea-N excretion by approximately 80%. Over the first 24 h of protein restriction, urinary sulfate excretion decreased more than urea-N excretion for both the normal and IDDM subjects. Under conditions of a high prior protein intake, fed-state sulfate production was normal for the IDDM subjects; protein restriction reduced fed-state sulfate production by 51% (normal subjects) and 59% (IDDM subjects; not significant). We conclude that whole body SAA metabolism is normal in conventionally treated IDDM before and after dietary protein restriction. SAA catabolism, as measured by fed-state sulfate production, may be a convenient and useful method to determine the extent of whole body protein dysregulation in IDDM.     

The Phosphate-Binding Protein of Escherichia coli Is Not Essential for Pi-Regulated Expression of the pho Regulon

Disruption of pstS encoding the P(i)-binding protein in Escherichia coli generally leads to the constitutive expression of the pho regulon. We demonstrate that P(i)-controlled expression is restored when the activity of the P(i) transporter PitA or PitB is increased. Apparently, PstS is not an essential component of the signal transduction pathway.