Three different classes of aminotransferases evolved prephenate aminotransferase functionality in arogenate-competent microorganisms.

The aromatic amino acids phenylalanine and tyrosine represent essential sources of high value natural aromatic compounds for human health and industry. Depending on the organism, alternative routes exist for their synthesis. Phenylalanine and tyrosine are synthesized either via phenylpyruvate/4-hydroxyphenylpyruvate or via arogenate. In arogenate-competent microorganisms, an aminotransferase is required for the transamination of prephenate into arogenate, but the identity of the genes is still unknown. We present here the first identification of prephenate aminotransferases (PATs) in seven arogenate-competent microorganisms and the discovery that PAT activity is provided by three different classes of aminotransferase, which belong to two different fold types of pyridoxal phosphate enzymes: an aspartate aminotransferase subgroup 1ß in tested α- and ß-proteobacteria, a branched-chain aminotransferase in tested cyanobacteria, and an N-succinyldiaminopimelate aminotransferase in tested actinobacteria and in the ß-proteobacterium Nitrosomonas europaea. Recombinant PAT enzymes exhibit high activity toward prephenate, indicating that the corresponding genes encode bona fide PAT. PAT functionality was acquired without other modification of substrate specificity and is not a general catalytic property of the three classes of aminotransferases.

HIF-1 attenuates Ref-1 expression in endothelial cells: reversal by siRNA and inhibition of geranylgeranylation.

Redox factor-1 (Ref-1), a multifunctional protein with DNA repairing activities, plays a cytoprotective function by post-translational redox modification of numerous transcription factors, including hypoxia inducible factor-1 (HIF-1). In the present study, activation of HIF-1 by hypoxia and dimethyloxaloylglycine (DMOG), a hypoxia mimic, diminished Ref-1 mRNA and protein expression in human microvascular endothelial cells (HMEC-1). Similarly, adenoviral delivery of the stabilized form of HIF-1alpha decreased Ref-1 mRNA and protein levels. Accordingly, HIF-1alpha siRNA abolished the hypoxia-induced inhibition of Ref-1 expression, indicating the role of HIF-1 in down-regulation of Ref-1. Also, translocation of Ref-1 from nucleus to cytoplasm after HIF-1 activation was noted. Interestingly, we observed the restoration of Ref-1 expression in hypoxia by pharmacologically relevant doses of atorvastatin. This effect was dependent on the inhibition of protein geranylgeranylation, but not farnesylation, as only the inhibitor of the former but not the latter prenylation step restored the Ref-1 expression. The regulation of Ref-1 by statins may be considered as a novel mechanism of their beneficial effects on endothelium.

Identification of critical residues in the active site of porcine membrane-bound aminopeptidase P.

The membrane-bound form of mammalian aminopeptidase P (AP-P; EC 3.4. 11.9) is a mono-zinc-containing enzyme that lacks any of the typical metal binding motifs found in other zinc metalloproteases. To identify residues involved in metal binding and catalysis, sequence and structural information was used to align the sequence of porcine membrane-bound AP-P with other members of the peptidase clan MG, including Escherichia coli AP-P and methionyl aminopeptidases. Residues predicted to be critical for activity were mutated and the resultant proteins were expressed in COS-1 cells. Immunoelectrophoretic blot analysis was used to compare the levels of expression of the mutant proteins, and their ability to hydrolyze bradykinin and Gly-Pro-hydroxyPro was assessed. Asp449, Asp460, His523, Glu554, and Glu568 are predicted to serve as metal ion ligands in the active site, and mutagenesis of these residues resulted in fully glycosylated proteins that were catalytically inactive. Mutation of His429 and His532 also resulted in catalytically inactive proteins, and these residues, by analogy with E. coli AP-P, are likely to play a role in shuttling protons during catalysis. These studies indicate that mammalian membrane-bound AP-P has an active-site configuration similar to that of other members of the peptidase clan MG, which is compatible with either a dual metal ion model or a single metal ion in the active site. The latter model is consistent, however, with the known metal stoichiometry of both the membrane-bound and cytosolic forms of AP-P and with a recently proposed model for methionyl aminopeptidase.

Evaluation of the role of specific acidic amino acid residues in electron transfer between the flavodoxin and cytochrome c3 from Desulfovibrio vulgaris.

A hypothetical model for electron transfer complex between cytochrome c3 and the flavodoxin from the sulfate-reducing bacteria Desulfovibrio vulgaris has been proposed, based on electrostatic potential field calculations and NMR data [Stewart, D. E., LeGall, J. , Moura, I., Moura, J. J. G., Peck, H. D., Jr., Xavier, A. V., Weiner, P. K., & Wampler, J. E. (1988) Biochemistry 27, 2444-2450]. This modeled complex relies primarily on the formation of five ion pairs between lysine residues of the cytochrome and acidic residues surrounding the flavin mononucleotide cofactor of the flavodoxin. In this study, the role of several acidic residues of the flavodoxin in the formation of this complex and in electron transfer between these two proteins was evaluated. A total of 17 flavodoxin mutants were studied in which 10 acidic amino acids--Asp62, Asp63, Glu66, Asp69, Asp70, Asp95, Glu99, Asp106, Asp127, and Asp129--had been permanently neutralized either individually or in various combinations by substitution with their amide amino acid equivalent (i.e., asparate to asparagine, glutamate to glutamine) through site-directed mutagenesis. The kinetic data for the transfer of electrons from reduced cytochrome c3 to the various flavodoxin mutants do not conform well to a simple bimolecular mechanism involving the formation of an intermediate electron transfer complex. Instead, a minimal electron transfer mechanism is proposed in which an initial complex is formed that is stabilized by intermolecular electrostatic interactions but is relatively inefficient in terms of electron transfer. This step is followed by a rate-limiting reorganization of that complex leading to efficient electron transfer. The apparent rate of this reorganization step was enhanced by the disruption of the initial electrostatic interactions through the neutralization of certain acidic amino acid residues leading to faster overall observed electron transfer rates at low ionic strengths. Of the five acidic residues involved in ion pairing in the modeled complex proposed by Stewart et al. (1988), the kinetic data strongly implicate Asp62, Glu66, and Asp95 in the formation of the electrostatic interactions that control electron transfer. Less certainty is provided by this study for the involvement of Asp69 and Asp129, although the data do not exclude their participation. It was not possible to determine whether the modeled complex represents the optimal configuration for electron transfer obtained after the reorganization step or actually represents the initial complex. The data do provide evidence for the importance of electrostatic interactions in electron transfer between these two proteins and for the existence of alternative binding modes involving acidic residues on the surface of the flavodoxin other than those proposed in that model.

Role of reductase domain cluster 1 acidic residues in neuronal nitric-oxide synthase. Characterization of the FMN-FREE enzyme.

The nNOS reductase domain is homologous to cytochrome P450 reductase, which contains two conserved clusters of acidic residues in its FMN module that play varied roles in its electron transfer reactions. To study the role of nNOS reductase domain cluster 1 acidic residues, we mutated two conserved acidic (Asp(918) and Glu(919)) and one conserved aromatic residue (Phe(892)), and investigated the effect of each mutation on flavin binding, conformational change, electron transfer reactions, calmodulin regulation, and catalytic activities. Each mutation destabilized FMN binding without significantly affecting other aspects including substrate, cofactor or calmodulin binding, or catalytic activities upon FMN reconstitution, indicating the mutational effect was restricted to the FMN module. Characterization of the FMN-depleted mutants showed that bound FMN was essential for reduction of the nNOS heme or cytochrome c, but not for ferricyanide or dichlorophenolindolphenol, and established that the electron transfer path in nNOS is NADPH to FAD to FMN to heme. Steady-state and stopped-flow kinetic analysis revealed a novel role for bound FMN in suppressing FAD reduction by NADPH. The suppression could be relieved either by FMN removal or calmodulin binding. Calmodulin binding induced a conformational change that was restricted to the FMN module. This increased the rate of FMN reduction and triggered electron transfer to the heme. We propose that the FMN module of nNOS is the key positive or negative regulator of electron transfer at all points in nNOS. This distinguishes nNOS from other related flavoproteins, and helps explain the mechanism of calmodulin regulation.