Rhodanese-Fold Containing Proteins in Humans: Not Just Key Players in Sulfur Trafficking.

The Rhodanese-fold is a ubiquitous structural domain present in various protein subfamilies associated with different physiological functions or pathophysiological conditions in humans. Proteins harboring a Rhodanese domain are diverse in terms of domain architecture, with some representatives exhibiting one or several Rhodanese domains, fused or not to other structural domains. The most famous Rhodanese domains are catalytically active, thanks to an active-site loop containing an essential cysteine residue which allows for catalyzing sulfur transfer reactions involved in sulfur trafficking, hydrogen sulfide metabolism, biosynthesis of molybdenum cofactor, thio-modification of tRNAs or protein urmylation. In addition, they also catalyse phosphatase reactions linked to cell cycle regulation, and recent advances proposed a new role into tRNA hydroxylation, illustrating the catalytic versatility of Rhodanese domain. To date, no exhaustive analysis of Rhodanese containing protein equipment from humans is available. In this review, we focus on structural and biochemical properties of human-active Rhodanese-containing proteins, in order to provide a picture of their established or putative key roles in many essential biological functions.

Aerobic Conditions and Endogenous Reactive Oxygen Species Reduce the Production of Infectious MS2 Phage by Escherichia coli.

Most of the defective/non-infectious enteric phages and viruses that end up in wastewater originate in human feces. Some of the causes of this high level of inactivity at the host stage are unknown. There is a significant gap between how enteric phages are environmentally transmitted and how we might design molecular tools that would only detect infectious ones. Thus, there is a need to explain the low proportion of infectious viral particles once replicated. By analyzing lysis plaque content, we were able to confirm that, under aerobic conditions, Escherichia coli produce low numbers of infectious MS2 phages (I) than the total number of phages indicated by the genome copies (G) with an I/G ratio of around 2%. Anaerobic conditions of replication and ROS inhibition increase the I/G ratio to 8 and 25%, respectively. These data cannot only be explained by variations in the total numbers of MS2 phages produced or in the metabolism of E. coli. We therefore suggest that oxidative damage impacts the molecular replication and assembly of MS2 phages.

CRDSAT Generated by pCARGHO: A New Efficient Lectin-Based Affinity Tag Method for Safe, Simple, and Low-Cost Protein Purification.

Purification of recombinant proteins remains a bottleneck for downstream processing. The authors engineered a new galectin 3 truncated form (CRDSAT ), functionally and structurally characterized, with preserved solubility and lectinic activity. Taking advantage of these properties, the authors designed an expression vector (pCARGHO), suitable for CRDSAT -tagged protein expression in prokaryotes. CRDSAT binds to lactose-Sepharose with a high specificity and facilitates solubilization of fusion proteins. This tag is structurally stable and can be easily removed from fusion proteins using TEV protease. Furthermore, due to their basic isoelectric point (pI), CRDSAT , and TEV are efficiently eliminated using cationic exchange chromatography. When pI of the protein of interest (POI) and CRDSAT are close, other chromatographic methods are successfully tested. Using CRDSAT tag, the authors purified several proteins from prokaryote and eukaryote origin and demonstrated as examples, the preservation of both Escherichia coli Thioredoxin 1 and human CDC25Bcd activities. Overall, yields of proteins obtained after tag removal are about 5-50 mg per litre of bacterial culture. Our purification method displays various advantages described herein that may greatly interest academic laboratories, biotechnology, and pharmaceutical companies.

Catalytic properties of a bacterial acylating acetaldehyde dehydrogenase: evidence for several active oligomeric states and coenzyme A activation upon binding.

Until the last decade, two unrelated aldehyde dehydrogenase (ALDH) superfamilies, i.e. the phosphorylating and non-phosphorylating superfamilies, were known to catalyze the oxidation of aldehydes to activated or non-activated acids. However, a third one was discovered by the crystal structure of a bifunctional enzyme 4-hydroxy-2-ketovalerate aldolase/acylating acetaldehyde dehydrogenase (DmpFG) from Pseudomonas sp. strain CF600 (Manjasetty et al., Proc. Natl. Acad. Sci. USA 100 (2003) 6992-6997). Indeed, DmpF exhibits a non-phosphorylating CoA-dependent ALDH activity, but is structurally related to the phosphorylating superfamily. In this study, we undertook the characterization of the catalytic and structural properties of MhpEF from Escherichia coli, an ortholog of DmpFG in which MhpF converts acetaldehyde, produced by the cleavage of 4-hydroxy-2-ketovalerate by MhpE, into acetyl-CoA. The kinetic data obtained under steady-state and pre-steady-state conditions show that the aldehyde dehydrogenase, MhpF, is active as a monomer, a unique feature relative to the phosphorylating and non-phosphorylating ALDH superfamilies. Our results also reveal that the catalytic properties of MhpF are not dependent on its oligomeric state, supporting the hypothesis of a structurally and catalytically independent entity. Moreover, the transthioesterification is shown to be rate-limiting and, when compared with a chemical model, its catalytic efficiency is increased 10(4)-fold. Therefore, CoA binding to MhpF increases its reactivity and optimizes its positioning relative to the thioacylenzyme intermediate, thus enabling the formation of an efficient deacylation complex.

Retinoic acid biosynthesis catalyzed by retinal dehydrogenases relies on a rate-limiting conformational transition associated with substrate recognition.

Retinoic acid (RA), a metabolite of vitamin A, exerts pleiotropic effects throughout life in vertebrate organisms. Thus, RA action must be tightly regulated through the coordinated action of biosynthetic and degrading enzymes. The last step of retinoic acid biosynthesis is irreversibly catalyzed by the NAD-dependent retinal dehydrogenases (RALDH), which are members of the aldehyde dehydrogenase (ALDH) superfamily. Low intracellular retinal concentrations imply efficient substrate molecular recognition to ensure high affinity and specificity of RALDHs for retinal. This study addresses the molecular basis of retinal recognition in human ALDH1A1 (or RALDH1) and rat ALDH1A2 (or RALDH2), through the comparison of the catalytic behavior of retinal analogs and use of the fluorescence properties of retinol. We show that, in contrast to long chain unsaturated substrates, the rate-limiting step of retinal oxidation by RALDHs is associated with acylation. Use of the fluorescence resonance energy transfer upon retinol interaction with RALDHs provides evidence that retinal recognition occurs in two steps: binding into the substrate access channel, and a slower structural reorganization with a rate constant of the same magnitude as the kcat for retinal oxidation: 0.18 vs. 0.07 and 0.25 vs. 0.1 s(-1) for ALDH1A1 and ALDH1A2, respectively. This suggests that the conformational transition of the RALDH-retinal complex significantly contributes to the rate-limiting step that controls the kinetics of retinal oxidation, as a prerequisite for the formation of a catalytically competent Michaelis complex. This conclusion is consistent with the general notion that structural flexibility within the active site of ALDH enzymes has been shown to be an integral component of catalysis.

Adenine binding mode is a key factor in triggering the early release of NADH in coenzyme A-dependent methylmalonate semialdehyde dehydrogenase.

Structural dynamics associated with cofactor binding have been shown to play key roles in the catalytic mechanism of hydrolytic NAD(P)-dependent aldehyde dehydrogenases (ALDH). By contrast, no information is available for their CoA-dependent counterparts. We present here the first crystal structure of a CoA-dependent ALDH. The structure of the methylmalonate semialdehyde dehydrogenase (MSDH) from Bacillus subtilis in binary complex with NAD(+) shows that, in contrast to what is observed for hydrolytic ALDHs, the nicotinamide ring is well defined in the electron density due to direct and H(2)O-mediated hydrogen bonds with the carboxamide. The structure also reveals that a conformational isomerization of the NMNH is possible in MSDH, as shown for hydrolytic ALDHs. Finally, the adenine ring is substantially more solvent-exposed, a result that could be explained by the presence of a Val residue at position 229 in helix α(F) that reduces the depth of the binding pocket and the absence of Gly-225 at the N-terminal end of helix α(F). Substitution of glycine for Val-229 and/or insertion of a glycine residue at position 225 resulted in a significant decrease of the rate constant associated with the dissociation of NADH from the NADH/thioacylenzyme complex, thus demonstrating that the weaker stabilization of the adenine ring is a key factor in triggering the early NADH release in the MSDH-catalyzed reaction. This study provides for the first time structural insights into the mechanism whereby the cofactor binding mode is responsible at least in part for the different kinetic behaviors of the hydrolytic and CoA-dependent ALDHs.

Methylmalonate-semialdehyde dehydrogenase from Bacillus subtilis: substrate specificity and coenzyme A binding.

Methylmalonate-semialdehyde dehydrogenase (MSDH) belongs to the CoA-dependent aldehyde dehydrogenase subfamily. It catalyzes the NAD-dependent oxidation of methylmalonate semialdehyde (MMSA) to propionyl-CoA via the acylation and deacylation steps. MSDH is the only member of the aldehyde dehydrogenase superfamily that catalyzes a ß-decarboxylation process in the deacylation step. Recently, we demonstrated that the ß-decarboxylation is rate-limiting and occurs before CoA attack on the thiopropionyl enzyme intermediate. Thus, this prevented determination of the transthioesterification kinetic parameters. Here, we have addressed two key aspects of the mechanism as follows: 1) the molecular basis for recognition of the carboxylate of MMSA; and 2) how CoA binding modulates its reactivity. We substituted two invariant arginines, Arg-124 and Arg-301, by Leu. The second-order rate constant for the acylation step for both mutants was decreased by at least 50-fold, indicating that both arginines are essential for efficient MMSA binding through interactions with the carboxylate group. To gain insight into the transthioesterification, we substituted MMSA with propionaldehyde, as both substrates lead to the same thiopropionyl enzyme intermediate. This allowed us to show the following: 1) the pK(app) of CoA decreases by ∼3 units upon binding to MSDH in the deacylation step; and 2) the catalytic efficiency of the transthioesterification is increased by at least 10(4)-fold relative to a chemical model. Moreover, we observed binding of CoA to the acylation complex, supporting a CoA-binding site distinct from that of NAD(H).

Stabilization and conformational isomerization of the cofactor during the catalysis in hydrolytic ALDHs.

Over the past 15 years, mechanistic and structural aspects were studied extensively for hydrolytic ALDHs. One the most striking feature of nearly all X-ray structures of binary ALDH-NAD(P)(+) complexes is the great conformational flexibility of the NMN moiety of the NAD(P)(+), in particular of the nicotinamide ring. However, the fact that the acylation step is efficient in GAPN (non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase) from Streptococcus mutans and in other hydrolytic ALDHs implies an optimal positioning of the nicotinamide ring relative to the hemithioacetal intermediate within the ternary complex to allow an efficient and stereospecific hydride transfer. Another key aspect of the chemical mechanism of this ALDH family is the requirement for the reduced NMN (NMNH) to move away from the initial position of the NMN for adequate positioning and activation of the deacylating water molecule by invariant E268 for completion of the reaction. In recent years, significant efforts have been made to characterize structural and molecular factors involved in the stabilization of the NMN moiety of the cofactor during the acylation step and to provide structural evidence of conformational isomerization of the cofactor during the catalytic cycle of hydrolytic ALDHs. The results presented here will be discussed for their relevance to the two-step catalytic mechanism and from an evolutionary viewpoint.

Invariant Thr244 is essential for the efficient acylation step of the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans.

One of the most striking features of several X-ray structures of CoA-independent ALDHs (aldehyde dehydrogenases) in complex with NAD(P) is the conformational flexibility of the NMN moiety. However, the fact that the rate of the acylation step is high in GAPN (non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase) from Streptococcus mutans implies an optimal positioning of the nicotinamide ring relative to the hemithioacetal intermediate within the ternary GAPN complex to allow an efficient and stereospecific hydride transfer. Substitutions of serine for invariant Thr244 and alanine for Lys178 result in a drastic decrease of the efficiency of hydride transfer which becomes rate-limiting. The crystal structure of the binary complex T244S GAPN-NADP shows that the absence of the beta-methyl group leads to a well-defined conformation of the NMN part, including the nicotinamide ring, clearly different from that depicted to be suitable for an efficient hydride transfer in the wild-type. The approximately 0.6-unit increase in pK(app) of the catalytic Cys302 observed in the ternary complex for both mutated GAPNs is likely to be due to a slight difference in positioning of the nicotinamide ring relative to Cys302 with respect to the wild-type ternary complex. Taken together, the data support a critical role of the Thr244 beta-methyl group, held in position through a hydrogen-bond interaction between the Thr244 beta-hydroxy group and the epsilon-amino group of Lys178, in permitting the nicotinamide ring to adopt a conformation suitable for an efficient hydride transfer during the acylation step for all the members of the CoA-independent ALDH family.

The first crystal structure of a thioacylenzyme intermediate in the ALDH family: new coenzyme conformation and relevance to catalysis.

Crystal structures of several members of the nonphosphorylating CoA-independent aldehyde dehydrogenase (ALDH) family have shown that the peculiar binding mode of the cofactor to the Rossmann fold results in a conformational flexibility for the nicotinamide moiety of the cofactor. This has been hypothesized to constitute an essential feature of the catalytic mechanism because the conformation of the cofactor required for the acylation step is not appropriate for the deacylation step. In the present study, the structure of a reaction intermediate of the E268A-glyceraldehyde 3-phosphate dehydrogenase (GAPN) from Streptococcus mutans, obtained by soaking the crystals of the enzyme/NADP complex with the natural substrate, is reported. The substrate is bound covalently in the four monomers and presents the geometric characteristics expected for a thioacylenzyme intermediate. Control experiments assessed that reduction of the coenzyme has occurred within the crystal. The structure reveals that reduction of the cofactor upon acylation leads to an extensive motion of the nicotinamide moiety with a flip of the reduced pyridinium ring away from the active site without significant changes of the protein structure. This event positions the reduced nicotinamide moiety in a pocket that likely constitutes the exit door for NADPH. Arguments are provided that the structure reported here constitutes a reasonable picture of the first thioacylenzyme intermediate characterized thus far in the ALDH family and that the position of the reduced nicotinamide moiety observed in GAPN is the one suitable for the deacylation step within all of the nonphosphorylating CoA-independent ALDH family.

Mechanistic characterization of the MSDH (methylmalonate semialdehyde dehydrogenase) from Bacillus subtilis.

Homotetrameric MSDH (methylmalonate semialdehyde dehydrogenase) from Bacillus subtilis catalyses the NAD-dependent oxidation of MMSA (methylmalonate semialdehyde) and MSA (malonate semialdehyde) into PPCoA (propionyl-CoA) and acetyl-CoA respectively via a two-step mechanism. In the present study, a detailed mechanistic characterization of the MSDH-catalysed reaction has been carried out. The results suggest that NAD binding elicits a structural imprinting of the apoenzyme, which explains the marked lag-phase observed in the activity assay. The enzyme also exhibits a half-of-the-sites reactivity, with two subunits being active per tetramer. This result correlates well with the presence of two populations of catalytic Cys302 in both the apo- and holo-enzymes. Binding of NAD causes a decrease in reactivity of the two Cys302 residues belonging to the two active subunits and a pKapp shift from approx. 8.8 to 8.0. A study of the rate of acylation as a function of pH revealed a decrease in the pKapp of the two active Cys302 residues to approx. 5.5. Taken to-gether, these results support a sequential Cys302 activation process with a pKapp shift from approx. 8.8 in the apo-form to 8.0 in the binary complex and finally to approx. 5.5 in the ternary complex. The rate-limiting step is associated with the b-decarboxylation process which occurs on the thioacylenzyme intermediate after NADH release and before transthioesterification. These data also indicate that bicarbonate, the formation of which is enzyme-catalysed, is the end-product of the reaction.

Expression, purification, crystallization and preliminary X-ray diffraction data of methylmalonate-semialdehyde dehydrogenase from Bacillus subtilis.

Methylmalonate-semialdehyde dehydrogenase from Bacillus subtilis was cloned and overexpressed in Escherichia coli. Suitable crystals for X-ray diffraction experiments were obtained by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 195.2, b = 192.5, c = 83.5 A, and contain one tetramer per asymmetric unit. X-ray diffraction data were collected to 2.5 A resolution using a synchrotron-radiation source. The crystal structure was solved by the molecular-replacement method.

Flavin radicals, conformational sampling and robust design principles in interprotein electron transfer: the trimethylamine dehydrogenase-electron-transferring flavoprotein complex.

TMADH (trimethylamine dehydrogenase) is a complex iron-sulphur flavoprotein that forms a soluble electron-transfer complex with ETF (electron-transferring flavoprotein). The mechanism of electron transfer between TMADH and ETF has been studied using stopped-flow kinetic and mutagenesis methods, and more recently by X-ray crystallography. Potentiometric methods have also been used to identify key residues involved in the stabilization of the flavin radical semiquinone species in ETF. These studies have demonstrated a key role for 'conformational sampling' in the electron-transfer complex, facilitated by two-site contact of ETF with TMADH. Exploration of three-dimensional space in the complex allows the FAD of ETF to find conformations compatible with enhanced electronic coupling with the 4Fe-4S centre of TMADH. This mechanism of electron transfer provides for a more robust and accessible design principle for interprotein electron transfer compared with simpler models that invoke the collision of redox partners followed by electron transfer. The structure of the TMADH-ETF complex confirms the role of key residues in electron transfer and molecular assembly, originally suggested from detailed kinetic studies in wild-type and mutant complexes, and from molecular modelling.

Extensive conformational sampling in a ternary electron transfer complex.

Here we report the crystal structures of a ternary electron transfer complex showing extensive motion at the protein interface. This physiological complex comprises the iron-sulfur flavoprotein trimethylamine dehydrogenase and electron transferring flavoprotein (ETF) from Methylophilus methylotrophus. In addition, we report the crystal structure of free ETF. In the complex, electron density for the FAD domain of ETF is absent, indicating high mobility. Positions for the FAD domain are revealed by molecular dynamics simulation, consistent with crystal structures and kinetic data. A dual interaction of ETF with trimethylamine dehydrogenase provides for dynamical motion at the protein interface: one site acts as an anchor, thereby allowing the other site to sample a large range of interactions, some compatible with rapid electron transfer. This study establishes the role of conformational sampling in multi-domain redox systems, providing insight into electron transfer between ETFs and structurally distinct redox partners.

Electron transfer and conformational change in complexes of trimethylamine dehydrogenase and electron transferring flavoprotein.

The trimethylamine dehydrogenase-electron transferring flavoprotein (TMADH.ETF) electron transfer complex has been studied by fluorescence and absorption spectroscopies. These studies indicate that a series of conformational changes occur during the assembly of the TMADH.ETF electron transfer complex and that the kinetics of assembly observed with mutant TMADH (Y442F/L/G) or ETF (alpha R237A) complexes are much slower than are the corresponding rates of electron transfer in these complexes. This suggests that electron transfer does not occur in the thermodynamically most favorable state (which takes too long to form), but that one or more metastable states (which are formed more rapidly) are competent in transferring electrons from TMADH to ETF. Additionally, fluorescence spectroscopy studies of the TMADH.ETF complex indicate that ETF undergoes a stable conformational change (termed structural imprinting) when it interacts transiently with TMADH to form a second, distinct, structural form. The mutant complexes compromise imprinting of ETF, indicating a dependence on the native interactions present in the wild-type complex. The imprinted form of semiquinone ETF exhibits an enhanced rate of electron transfer to the artificial electron acceptor, ferricenium. Overall molecular conformations as probed by small-angle x-ray scattering studies are indistinguishable for imprinted and non-imprinted ETF, suggesting that changes in structure likely involve confined reorganizations within the vicinity of the FAD. Our results indicate a series of conformational events occur during the assembly of the TMADH.ETF electron transfer complex, and that the properties of electron transfer proteins can be affected lastingly by transient interaction with their physiological redox partners. This may have significant implications for our understanding of biological electron transfer reactions in vivo, because ETF encounters TMADH at all times in the cell. Our studies suggest that caution needs to be exercised in extrapolating the properties of in vitro interprotein electron transfer reactions to those occurring in vivo.