Bradyrhizobium japonicum porphobilinogen synthase (B. japonicum PBGS) has been purified and characterized from an overexpression system in an Escherichia coli host (Chauhan, S., and O'Brian, M. R. (1995) J. Biol. Chem. 270, 19823-19827 Porphobilinogen synthase (PBGS, 1 also known as 5-aminolevulinate dehydratase, E.C. 4.2.1.24) is a metalloenzyme that catalyzes the asymmetric condensation of two molecules of 5-aminolevulinate (ALA) to form porphobilinogen as illustrated in Fig. 1 (1). This reaction is common to tetrapyrrole biosynthesis in all phyla (e.g. porphyrin, chlorophyll, corrin, and F430) and is essential for all cellular life. The PBGS octamer contains four active sites, each of which binds two molecules of ALA that have different chemical fates. Although many details of the reaction mechanism are not well established, it is known that there is a Schiff base formed between a universally conserved lysine and one of the two ALA molecules at the active site of the enzyme from all sources examined (2-6, 35). The universal Schiff base and high overall sequence conservation suggest that all PBGSs use a common catalytic mechanism (7). Nevertheless, there are well documented differences in metal ion requirements between evolutionarily divergent PBGS (for example, see Ref. 8).Until recently, PBGSs from various sources were generally described as requiring either Mg(II) or Zn(II). However, characterization of Escherichia coli PBGS demonstrates that this enzyme can bind both Zn(II) and Mg(II) at different sites and with different functions (9, 10). Based on studies of E. coli PBGS and mammalian PBGS, this laboratory proposed a model in which any given PBGS had up to three different types of divalent metal ion binding sites called A, B, and C. The function of the divalent ion in site A is to facilitate A-side ALA binding and reactivity (see Ref. 7). The B-site metal ion has not been found to be "essential" although it has been proposed to aid in the removal of a proton lost during porphobilinogen formation (11). Four A-site metal ions and four B-site metal ions bind to each octamer (12-14), equivalent to the stoichiometry of active sites. The C site metal is an allosteric activator that increases V max , decreases the K m for ALA, and decreases the K d for metal ions in sites A and B; its stoichiometry is 8/octamer (9). Table I The plant endosymbiot Bradyrhizobium japonicum produces a PBGS (B. japonicum PBGS) protein with a hybrid sequence in a putative metal binding domain of PBGS, shown in Fig. 2 (17, 18). To help explain the stoichiometry of sites A and B, 4 sites/homooctamer, this domain of each subunit has been proposed to provide ligands to either the A-site or B-site metal ions (13,14). The C site metal ions bind elsewhere in the sequence (20). 3 The sequence of B. japonicum PBGS in this region is different from those of types I, II, and III PBGS and may define a type IV PBGS. To explore the existence of a type IV PBGS, we purified and characterized B. japonicum PBGS cloned and expressed in E. coli (21). W...

Chromatin remodeling is of crucial importance during brain development. Pathogenic alterations of several chromatin remodeling ATPases have been implicated in neurodevelopmental disorders. We describe an index case with a de novo missense mutation in CHD3, identified during whole genome sequencing of a cohort of children with rare speech disorders. To gain a comprehensive view of features associated with disruption of this gene, we use a genotype-driven approach, collecting and characterizing 35 individuals with de novo CHD3 mutations and overlapping phenotypes. Most mutations cluster within the ATPase/helicase domain of the encoded protein. Modeling their impact on the three-dimensional structure demonstrates disturbance of critical binding and interaction motifs. Experimental assays with six of the identified mutations show that a subset directly affects ATPase activity, and all but one yield alterations in chromatin remodeling. We implicate de novo CHD3 mutations in a syndrome characterized by intellectual disability, macrocephaly, and impaired speech and language.

Lysine 2,3-aminomutase from Clostridia catalyzes the interconversion of L-alpha-lysine with L-beta-lysine. The purified enzyme contains iron-sulfur ([Fe-S]) clusters, pyridoxal phosphate, and Co(II) [Petrovich, R. M., Ruzicka, F. J., Reed, G. H., & Frey, P. A. (1991) J. Biol. Chem. 266, 7656-7660]. Enzymatic activity depends upon the presence and integrity of these cofactors. In addition, the enzyme is activated by S-adenosylmethionine, which participates in the transfer of a substrate hydrogen atom between carbon-3 of lysine and carbon-2 of beta-lysine [Moss, M., & Frey, P. A. (1987) J. Biol. Chem. 262, 14859-14862]. This paper describes the electron paramagnetic resonance (EPR) properties of the [Fe-S] clusters. Purified samples of the enzyme also contain low and variable levels of a stable radical. The radical spectrum is centered at g = 2.006 and is subject to inhomogeneous broadening at 10 K, with a p1/2 value of 550 +/- 100 microW. The low-temperature EPR spectrum of the [Fe-S] cluster is centered at g = 2.007 and undergoes power saturation at 10 K in a homogeneous manner, with a p1/2 of 15 +/- 2 mW. The signals are consistent with the formulation [4Fe-4S] and are adequately simulated by a rhombic spectrum, in which gxx = 2.027, gyy = 2.007, and gzz = 1.99. Treatment of the enzyme with reducing agents converts the cluster into an EPR-silent form. Oxidation of the purified enzyme by air or ferricyanide converts the [Fe-S] complex into a species with an EPR spectrum that is consistent with the formulation [3Fe-4S].(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescent proteins are an important tool that has become omnipresent in life sciences research. They are frequently used for localization of proteins and monitoring of cells [1], [2]. Green fluorescent protein (GFP) was the first and has been the most used fluorescent protein. Enhanced GFP (eGFP) was optimized from wild-type GFP for increased fluorescence yield and improved expression in mammalian systems [3]. Many GFP-like fluorescent proteins have been discovered, optimized or created, such as the red fluorescent protein TagRFP [4]. Fluorescent proteins are expressed colorless and immature and, for eGFP, the conversion to the fluorescent form, mature, is known to produce one equivalent of hydrogen peroxide (H2O2) per molecule of chromophore [5,6]. Even though it has been proposed that this process is non-catalytic and generates nontoxic levels of H2O2 [6], this study investigates the role of fluorescent proteins in generating free radicals and inducing oxidative stress in biological systems. Immature eGFP and TagRFP catalytically generate the free radical superoxide anion (O2•–) and H2O2 in the presence of NADH. Generation of the free radical O2•– and H2O2 by eGFP in the presence of NADH affects the gene expression of cells. Many biological pathways are altered, such as a decrease in HIF1α stabilization and activity. The biological pathways altered by eGFP are known to be implicated in the pathophysiology of many diseases associated with oxidative stress; therefore, it is critical that such experiments using fluorescent proteins are validated with alternative methodologies and the results are carefully interpreted. Since cells inevitably experience oxidative stress when fluorescent proteins are expressed, the use of this tool for cell labeling and in vivo cell tracing also requires validation using alternative methodologies.