Mutagenesis studies on the iron binding ligands of clavaminic acid synthase

Mutagenesis studies on conserved histidine residues identified as possible metal binding ligands in clavaminic acid synthase isozyme 2 were consistent with His-145 and His-280 acting as iron ligands, in support of crystallographic and previous mutagenesis studies. Mutagenesis of the four cysteines and a glutamine residue, conserved in both clavaminic acid synthase isozymes 1 and 2, demonstrated that none of these residues is essential for activity.     

Mechanism of the beta-ketoacyl synthase reaction catalyzed by the animal fatty acid synthase

The catalytic mechanism of the beta-ketoacyl synthase domain of the multifunctional fatty acid synthase has been investigated by a combination of mutagenesis, active-site titration, product analysis, and product inhibition. Neither the reactivity of the active-site Cys161 residue toward iodoacetamide nor the rate of unidirectional transfer of acyl moieties to Cys161 was significantly decreased by replacement of any of the conserved residues, His293, His331, or Lys326, with Ala. Decarboxylation of malonyl moieties in the fully-active Cys161Gln background generated equimolar amounts of acetyl-CoA and bicarbonate, rather than carbon dioxide, and was seriously compromised by replacement of any of the conserved basic residues. The ability of bicarbonate to inhibit decarboxylation of malonyl moieties in the Cys161Gln background was significantly reduced by replacement of His293 but less so by replacement of His331. The data are consistent with a reaction mechanism, in which the initial primer transfer reaction is promoted largely through a lowering of the pKa of the Cys161 thiol by a helix dipole effect and activation of the substrate thioester carbon atom by binding of the keto group in an oxyanion hole. The data also indicate that an activated water molecule is present at the active site that is required either for the rapid hydration of carbon dioxide, prior its release as bicarbonate or, alternatively, for an initial attack on the malonyl C3. In the alternative mechanism, a negatively-charged tetrahedral transition state could be generated, stabilized in part by interaction of His293 with the negatively charged oxygen at C3 and interaction of His331 with the negatively charged thioester carbonyl oxygen, that breaks down to generate bicarbonate directly. Finally, the carbanion at C2, attacks the electrophilic C1 of the primer, generating a second tetrahedral transition state, also stabilized through contacts with the oxyanion hole and His331, that breaks down to form the beta-ketoacyl-S-acyl carrier protein product.     

Insights into diterpene cyclization from structure of bifunctional abietadiene synthase from Abies grandis

Abietadiene synthase from Abies grandis (AgAS) is a model system for diterpene synthase activity, catalyzing class I (ionization-initiated) and class II (protonation-initiated) cyclization reactions. Reported here is the crystal structure of AgAS at 2.3 Å resolution and molecular dynamics simulations of that structure with and without active site ligands. AgAS has three domains (α, β, and γ). The class I active site is within the C-terminal α domain, and the class II active site is between the N-terminal γ and β domains. The domain organization resembles that of monofunctional diterpene synthases and is consistent with proposed evolutionary origins of terpene synthases. Molecular dynamics simulations were carried out to determine the effect of substrate binding on enzymatic structure. Although such studies of the class I active site do lead to an enclosed substrate-Mg²⁺ complex similar to that observed in crystal structures of related plant enzymes, it does not enforce a single substrate conformation consistent with the known product stereochemistry. Simulations of the class II active site were more informative, with observation of a well ordered external loop migration. This “loop-in” conformation not only limits solvent access but also greatly increases the number of conformational states accessible to the substrate while destabilizing the nonproductive substrate conformation present in the “loop-out” conformation. Moreover, these conformational changes at the class II active site drive the substrate toward the proposed transition state. Docked substrate complexes were further assessed with regard to the effects of site-directed mutations on class I and II activities.     

Studies on the substrate specificity of clavaminic acid synthase and associated enzymes

Fifteen structural analogues of proclavaminic acid have been synthesised and incubated with enzyme preparations from Streptomyces clavuligerus in order to test the substrate specificity of the enzymes responsible for converting monocyclic β-lactams to bicyclic β-lactams.     

Structural origins of the selectivity of the trifunctional oxygenase clavaminic acid synthase

Clavaminate synthase (CAS), a remarkable Fe(II)/2-oxoglutarate oxygenase, catalyzes three separate oxidative reactions in the biosynthesis of clavulanic acid, a clinically used inhibitor of serine beta-lactamases. The first CAS-catalyzed step (hydroxylation) is separated from the latter two (oxidative cyclization/desaturation) by the action of an amidinohydrolase. Here, we describe crystal structures of CAS in complex with Fe(II), 2-oxoglutarate (2OG) and substrates (N-alpha-acetyl-L-arginine and proclavaminic acid). They reveal how CAS catalyzes formation of the clavam nucleus, via a process unprecedented in synthetic organic chemistry, and suggest how it discriminates between substrates and controls reaction of its highly reactive ferryl intermediate. The presence of an unpredicted jelly roll beta-barrel core in CAS implies divergent evolution within the family of 2OG and related oxygenases. Comparison with other non-heme oxidases/oxygenases reveals flexibility in the position which dioxygen ligates to the iron, in contrast to the analogous heme-using enzymes.     

Mechanism of the physiological reaction catalyzed by tryptophan synthase from Escherichia coli

The physiological synthesis of L-tryptophan from indoleglycerol phosphate and L-serine catalyzed by the alpha 2 beta 2 bienzyme complex of tryptophan synthase requires spatial and dynamic cooperation between the two distant alpha and beta active sites. The carbanion of the adduct of L-tryptophan to pyridoxal phosphate accumulated during the steady state of the catalyzed reaction. Moreover, it was formed transiently and without a lag in single turnovers, and glyceraldehyde 3-phosphate was released only after formation of the carbanion. These and further data prove first that the affinity for indoleglycerol phosphate and its cleavage to indole in the alpha subunit are enhanced substantially by aminoacrylate bound to the beta subunit. This indirect activation explains why the turnover number of the physiological reaction is larger than that of the indoleglycerol phosphate cleavage reaction. Second, reprotonation of nascent tryptophan carbanion is rate limiting for overall tryptophan synthesis. Third, most of the indole generated in the active site of the alpha subunit is transferred directly to the active site of the beta subunit and only insignificant amounts pass through the solvent. Comparison of the single turnover rate constants with the known elementary rate constants of the partial reactions catalyzed by the alpha and beta active sites suggests that the cleavage reaction rather than the transfer of indole or its condensation with aminoacrylate is rate limiting for the formation of nascent tryptophan.     

Molecular characterization of farnesyl pyrophosphate synthase from Bacopa monniera by comparative modeling and docking studies

Farnesyl pyrophosphate synthase (FPS; EC 2.5.1.10) is a key enzyme in isoprenoid biosynthetic pathway and provides precursors for the biosynthesis of various pharmaceutically important metabolites. It catalyzes head to tail condensation of two isopentenyl pyrophosphate molecules with dimethylallyl pyrophosphate to form C15 compound farnesyl pyrophosphate. Recent studies have confirmed FPS as a molecular target of bisphosphonates for drug development against bone diseases as well as pathogens. Although large numbers of FPSs from different sources are known, very few protein structures have been reported till date. In the present study, FPS gene from medicinal plant Bacopa monniera (BmFPS) was characterized by comparative modeling and docking. Multiple sequence alignment showed two highly conserved aspartate rich motifs FARM and SARM (DDXXD). The 3-D model of BmFPS was generated based on structurally resolved FPS crystal information of Gallus gallus. The generated models were validated by various bioinformatics tools and the final model contained only α-helices and coils. Further, docking studies of modeled BmFPS with substrates and inhibitors were performed to understand the protein ligand interactions. The two Asp residues from FARM (Asp100 and Asp104) as well as Asp171, Lys197 and Lys262 were found to be important for catalytic activity. Interaction of nitrogen containing bisphosphonates (risedronate, alendronate, zoledronate and pamidronate) with modeled BmFPS showed competitive inhibition; where, apart from Asp (100, 104 and 171), Thr175 played an important role. The results presented here could be useful for designing of mutants for isoprenoid biosynthetic pathway engineering well as more effective drugs against osteoporosis and human pathogens.

Abbreviations

IPP - Isopentenyl Pyrophosphate, DMAPP - Dimethylallyl Pyrophosphate, GPP - Geranyl Pyrophosphate, FPP - FPPFarnesyl Pyrophosphate, DOPE - Discrete Optimized Protein Energy, BmFPS - Bacopa monniera Farnesyl Pyrophosphate Synthase, RMSD - Root Mean square Deviation, OPLS-AA - Optimized Potentials for Liquid Simulations- All Atom, FARM - First Aspartate Rich Motif, SARM - Second Aspartate Rich Motif.     

Insights into Zn2+ homeostasis in neurons from experimental and modeling studies

To understand the mechanisms of neuronal Zn2+ homeostasis better, experimental data obtained from cultured cortical neurons were used to inform a series of increasingly complex computational models. Total metals (inductively coupled plasma-mass spectrometry), resting metallothionein, (65)Zn2+ uptake and release, and intracellular free Zn2+ levels using ZnAF-2F were determined before and after neurons were exposed to increased Zn2+, either with or without the addition of a Zn2+ ionophore (pyrithione) or metal chelators [EDTA, clioquinol (CQ), and N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine]. Three models were tested for the ability to match intracellular free Zn2+ transients and total Zn2+ content observed under these conditions. Only a model that incorporated a muffler with high affinity for Zn2+, trafficking Zn2+ to intracellular storage sites, was able to reproduce the experimental results, both qualitatively and quantitatively. This "muffler model" estimated the resting intracellular free Zn2+ concentration to be 1.07 nM. If metallothionein were to function as the exclusive cytosolic Zn2+ muffler, the muffler model predicts that the cellular concentration required to match experimental data is greater than the measured resting concentration of metallothionein. Thus Zn2+ buffering in resting cultured neurons requires additional high-affinity cytosolic metal binding moieties. Added CQ, as low as 1 microM, was shown to selectively increase Zn2+ influx. Simulations reproduced these data by modeling CQ as an ionophore. We conclude that maintenance of neuronal Zn2+ homeostasis, when challenged with Zn2+ loads, relies heavily on the function of a high-affinity muffler, the characteristics of which can be effectively studied with computational models.     

Insights from modeling studies on how climate change affects invasive alien species geography

Climate change and biological invasions are threatening biodiversity and ecosystem services worldwide. It has now been widely acknowledged that climate change will affect biological invasions. A large number of studies have investigated predicted shifts and other changes in the geographic ranges of invasive alien species related to climate change using modeling approaches. Yet these studies have provided contradictory evidence, and no consensus has been reached. We conducted a systematic review of 423 modeling case studies included in 71 publications that have examined the predicted effects of climate change on those species. We differentiate the approaches used in these studies and synthesize their main results. Our results reaffirm the major role of climate change as a driver of invasive alien species distribution in the future. We found biases in the literature both regarding the taxa, toward plants and invertebrates, and the areas of the planet investigated. Despite these biases, we found for the plants and vertebrates studied that climate change will more frequently contribute to a decrease in species range size than an increase in the overall area occupied. This is largely due to oceans preventing terrestrial invaders from spreading poleward. In contrast, we found that the ranges of invertebrates and pathogens studied are more likely to increase following climate change. An important caveat to these findings is that researchers have rarely considered the effects of climate change on transport, introduction success, or the resulting impacts. We recommend closing these research gaps, and propose additional avenues for future investigations, as well as opportunities and challenges for managing invasions under climate change.     

Insights from modeling protein evolution with context-dependent mutation and asymmetric amino acid selection

We develop an approximate maximum likelihood method to estimate flanking nucleotide context-dependent mutation rates and amino acid exchange-dependent selection in orthologous protein-coding sequences and use it to analyze genome-wide coding sequence alignments from mammals and yeast. Allowing context-dependent mutation provides a better fit to coding sequence data than simpler (context-independent or CpG "hotspot") models and significantly affects selection parameter estimates. Allowing asymmetric (nonreciprocal) selection on amino acid exchanges gives a better fit than simple dN/dS or symmetric selection models. Relative selection strength estimates from our models show good agreement with independent estimates derived from human disease-causing and engineered mutations. Selection strengths depend on local protein structure, showing expected biophysical trends in helical versus nonhelical regions and increased asymmetry on polar-hydrophobic exchanges with increased burial. The more stringent selection that has previously been observed for highly expressed proteins is primarily concentrated in buried regions, supporting the notion that such proteins are under stronger than average selection for stability. Our analyses indicate that a highly parameterized model of mutation and selection is computationally tractable and is a useful tool for exploring a variety of biological questions concerning protein and coding sequence evolution.     

Mechanism of the reaction catalyzed by acyl-CoA: lysolecithin acyltransferase from rabbit lung. pH studies and chemical modification

This paper deals with the first attempt to elucidate the chemical mechanism of acyl-CoA: lysolecithin acyltransferase from rabbit lung, a key enzyme in the metabolism of lung surfactant. For this purpose, the pH dependence of kinetic constants as well as the chemical modification of the protein have been studied on a partially-purified preparation. From these experiments, the pKs on which the activity of the enzyme relies have been calculated, giving values of pK1 congruent to 5.5 and pK2 congruent to 10. Analysis of the effect of organic solvents on these pKs and the calculation of the enthalpies of ionization, together with the chemical modification experiments, lead to the conclusion that pK1 is due to an histidine residue, whereas pK2 arises from the amino group of the adenine ring of palmitoyl-CoA. Moreover, chemical modification demonstrated an essential cysteine. A tentative chemical mechanism, in accordance with these results, is proposed and it is hypothesized, in view of other results obtained in our laboratory and from the literature, that the chemical mechanism of acyl transfer to sn-2 position may be common to other enzymes of glycerolipid metabolism.     

High-pressure studies of the reaction mechanism of nitric-oxide synthase

Nitric-oxide synthase (NOS) generates nitric oxide from l-arginine in two reaction cycles with N(omega)-hydroxy-l-arginine as an obligate intermediate. Although much progress has been made in recent years in the elucidation of the reaction mechanism of NOS, many questions remain to be answered. The use of low temperature has been instrumental in the revelation of the mechanism of NO synthesis, particularly regarding the role of the cofactor 5,6,7,8-tetrahydrobopterin (BH4). High-pressure studies may be expected to be similarly useful, but have been very few so far. In this short review, we depict the present state of knowledge about the reaction mechanism of NO synthesis, and the role(s) BH4 plays in it. This exposition is followed by a summary of the results obtained thus far in high-pressure studies and of the conclusions that can be drawn from them.     

Substrate binding mode and reaction mechanism of undecaprenyl pyrophosphate synthase deduced from crystallographic studies

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes eight consecutive condensation reactions of farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to form a 55-carbon long-chain product. We previously reported the crystal structure of the apo-enzyme from Escherichia coli and the structure of UPPs in complex with sulfate ions (resembling pyrophosphate of substrate), Mg(2+), and two Triton molecules (product-like). In the present study, FPP substrate was soaked into the UPPs crystals, and the complex structure was solved. Based on the crystal structure, the pyrophosphate head group of FPP is bound to the backbone NHs of Gly29 and Arg30 as well as the side chains of Asn28, Arg30, and Arg39 through hydrogen bonds. His43 is close to the C2 carbon of FPP and may stabilize the farnesyl cation intermediate during catalysis. The hydrocarbon moiety of FPP is bound with hydrophobic amino acids including Leu85, Leu88, and Phe89, located on the alpha3 helix. The binding mode of FPP in cis-type UPPs is apparently different from that of trans-type and many other prenyltransferases which utilize Asprich motifs for substrate binding via Mg(2+). The new structure provides a plausible mechanism for the catalysis of UPPs.     

Monitoring the acetohydroxy acid synthase reaction and related carboligations by circular dichroism spectroscopy

Acetohydroxy acid synthase (AHAS) and related enzymes catalyze the production of chiral compounds [(S)-acetolactate, (S)-acetohydroxybutyrate, or (R)-phenylacetylcarbinol] from achiral substrates (pyruvate, 2-ketobutyrate, or benzaldehyde). The common methods for the determination of AHAS activity have shortcomings. The colorimetric method for detection of acyloins formed from the products is tedious and does not allow time-resolved measurements. The continuous assay for consumption of pyruvate based on its absorbance at 333 nm, though convenient, is limited by the extremely small extinction coefficient of pyruvate, which results in a low signal-to-noise ratio and sensitivity to interfering absorbing compounds. Here, we report the use of circular dichroism spectroscopy for monitoring AHAS activity. This method, which exploits the optical activity of reaction products, displays a high signal-to-noise ratio and is easy to perform both in time-resolved and in commercial modes. In addition to AHAS, we examined the determination of activity of glyoxylate carboligase. This enzyme catalyzes the condensation of two molecules of glyoxylate to chiral tartronic acid semialdehyde. The use of circular dichroism also identifies the product of glyoxylate carboligase as being in the (R) configuration.     

Mechanism of the reaction catalyzed by dehydroascorbate reductase from spinach chloroplasts.

Dehydroascorbate reductase (DHAR) reduces dehydroascorbate (DHA) to ascorbate with glutathione (GSH) as the electron donor. We analyzed the reaction mechanism of spinach chloroplast DHAR, which had a much higher reaction specificity for DHA than animal enzymes, using a recombinant enzyme expressed in Escherichia coli. Kinetic analysis suggested that the reaction proceeded by a bi-uni-uni-uni-ping-pong mechanism, in which binding of DHA to the free, reduced form of the enzyme was followed by binding of GSH. The Km value for DHA and the summed Km value for GSH were determined to be 53 +/- 12 micro m and 2.2 +/- 1.0 mm, respectively, with a turnover rate of 490 +/- 40 s-1. Incubation of 10 microm DHAR with 1 mm DHA and 10 microm GSH resulted in stable binding of GSH to the enzyme. Bound GSH was released upon reduction of the GSH-enzyme adduct by 2-mercaptoethanol, suggesting that the adduct is a reaction intermediate. Site-directed mutagenesis indicated that C23 in DHAR is indispensable for the reduction of DHA. The mechanism of catalysis of spinach chloroplast DHAR is proposed.     

Selenocysteine synthase from Escherichia coli. Analysis of the reaction sequence

The product of the selA gene, selenocysteine synthase, is a pyridoxal 5-phosphate-containing enzyme which catalyzes the conversion of seryl-tRNA(Sec UCA) into selenocysteyl-tRNA(Sec UCA). Reduction of the aldimine group of pyridoxal 5-phosphate inactivates the enzyme. When reacted with seryl-tRNA(Sec UCA) as sole substrate, pyruvate (and possibly also ammonia) is released; in the presence of a high concentration of potassium borohydride, alanyl-tRNA(Sec UCA) is formed from seryl-tRNA(Sec UCA). These results support the notion that the formyl group of pyridoxal phosphate forms a Schiff base with the alpha-amino group of L-serine with the subsequent 2,3-elimination of a water molecule and the generation of an aminoacrylyl-tRNA(Sec UCA) intermediate. ATP is not required for this reaction step, but it is necessary for the conversion of aminoacrylyl-tRNA into selenocysteyl-tRNA(Sec UCA) which, in addition, requires the SELD protein and reduced selenium. Selenocysteine synthase forms a stable complex with seryl-tRNA(Sec UCA) with one tRNA molecule bound per two 50-kDa monomers. The enzyme does not interact with serine-inserting tRNA species. Taken together, the results show that biosynthesis of selenocysteine takes place in the enzyme-bound state and involves the dehydration of L-serine esterified to tRNA in a first step formally followed by the 2,3-addition of HSe- which is provided by the SELD protein in an ATP-dependent reaction in the form of a reactive selenium donor molecule.     

Kinetic studies on the reaction catalysed by phosphofructokinase from Trypanosoma brucei.

The steady-state kinetics of the reaction catalysed by the bloodstream form of Trypanosoma brucei were studied at pH 6.7. In the presence of 50 mM-potassium phosphate buffer, the apparent co-operativity with respect to fructose 6-phosphate and the non-linear relationship between initial velocity and enzyme concentration, which were found when the enzyme was assayed in 50 mM-imidazole buffer [Cronin & Tipton (1985) Biochem. J. 227, 113-124], are not evident. Studies on the variations of the initial rate with changing concentrations of MgATP and fructose 6-phosphate, the product inhibition by fructose 1,6-bisphosphate and the effects of the alternative substrate ITP were consistent with an ordered reaction pathway, in which MgATP binds to the enzyme before fructose 6-phosphate, and fructose 1,6-bisphosphate is the first product to dissociate from the ternary complex.