Assembly of PGAM5 into Multimeric Complexes Provides a Mechanism for Allosteric Regulation of Phosphatase Activity.

Phosphoglycerate mutase family member 5 (PGAM5) is a serine/threonine phosphatase that has been localized to both inner and outer mitochondrial membranes. PGAM5 has been suggested to regulate multiple aspects of mitochondrial dynamics, including fission/fusion and mitophagy, through phosphatase-dependent and phosphatase-independent mechanisms. Understanding how the phosphatase activity of PGAM5 is regulated will provide new insight into signaling mechanisms that link changes in cell physiology with mitochondrial function. In this chapter, we describe methods for obtaining both multimeric and dimeric complexes of PGAM5 and for characterizing their kinetic properties. The ability to purify different PGAM5 complexes and to characterize their kinetic properties will enable detailed biophysical studies of the quaternary structures of the various PGAM5-containing complexes. The phosphatase activity of different PGAM5 complexes varies over three orders of magnitude. We suggest that the ability to generate PGAM5 complexes that have a wide range of phosphatase activities will facilitate screens to identify small molecules that modulate the phosphatase activity of PGAM5.

The structure of glucose-1-phosphate thymidylyltransferase from Mycobacterium tuberculosis reveals the location of an essential magnesium ion in the RmlA-type enzymes.

Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, continues to be a major threat to populations worldwide. Whereas the disease is treatable, the drug regimen is arduous at best with the use of four antimicrobials over a six-month period. There is clearly a pressing need for the development of new therapeutics. One potential target for structure-based drug design is the enzyme RmlA, a glucose-1-phosphate thymidylyltransferase. This enzyme catalyzes the first step in the biosynthesis of l-rhamnose, which is a deoxysugar critical for the integrity of the bacterium's cell wall. Here, we report the X-ray structures of M. tuberculosis RmlA in complex with either dTTP or dTDP-glucose to 1.6 Å and 1.85 Å resolution, respectively. In the RmlA/dTTP complex, two magnesium ions were observed binding to the nucleotide, both ligated in octahedral coordination spheres. In the RmlA/dTDP-glucose complex, only a single magnesium ion was observed. Importantly, for RmlA-type enzymes with known three-dimensional structures, not one model shows the position of the magnesium ion bound to the nucleotide-linked sugar. As such, this investigation represents the first direct observation of the manner in which a magnesium ion is coordinated to the RmlA product and thus has important ramifications for structure-based drug design. In the past, molecular modeling procedures have been employed to derive a three-dimensional model of the M. tuberculosis RmlA for drug design. The X-ray structures presented herein provide a superior molecular scaffold for such endeavors in the treatment of one of the world's deadliest diseases.

Structural and Biochemical Investigation of PglF from Campylobacter jejuni Reveals a New Mechanism for a Member of the Short Chain Dehydrogenase/Reductase Superfamily.

Within recent years it has become apparent that protein glycosylation is not limited to eukaryotes. Indeed, in Campylobacter jejuni, a Gram-negative bacterium, more than 60 of its proteins are known to be glycosylated. One of the sugars found in such glycosylated proteins is 2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyranose, hereafter referred to as QuiNAc4NAc. The pathway for its biosynthesis, initiating with UDP-GlcNAc, requires three enzymes referred to as PglF, PglE, and PlgD. The focus of this investigation is on PglF, an NAD+-dependent sugar 4,6-dehydratase known to belong to the short chain dehydrogenase/reductase (SDR) superfamily. Specifically, PglF catalyzes the first step in the pathway, namely, the dehydration of UDP-GlcNAc to UDP-2-acetamido-2,6-dideoxy-α-d-xylo-hexos-4-ulose. Most members of the SDR superfamily contain a characteristic signature sequence of YXXXK where the conserved tyrosine functions as a catalytic acid or a base. Strikingly, in PglF, this residue is a methionine. Here we describe a detailed structural and functional investigation of PglF from C. jejuni. For this investigation five X-ray structures were determined to resolutions of 2.0 Å or better. In addition, kinetic analyses of the wild-type and site-directed variants were performed. On the basis of the data reported herein, a new catalytic mechanism for a SDR superfamily member is proposed that does not require the typically conserved tyrosine residue.

Biochemical Characterization of WbkC, an N-Formyltransferase from Brucella melitensis.

It has become increasingly apparent within the last several years that unusual N-formylated sugars are often found on the O-antigens of such Gram negative pathogenic organisms as Francisella tularensis, Campylobacter jejuni, and Providencia alcalifaciens, among others. Indeed, in some species of Brucella, for example, the O-antigen contains 1,2-linked 4-formamido-4,6-dideoxy-α-d-mannosyl groups. These sugars, often referred to as N-formylperosamine, are synthesized in pathways initiating with GDP-mannose. One of the enzymes required for the production of N-formylperosamine, namely, WbkC, was first identified in 2000 and was suggested to function as an N-formyltransferase. Its biochemical activity was never experimentally verified, however. Here we describe a combined structural and functional investigation of WbkC from Brucella melitensis. Four high resolution X-ray structures of WbkC were determined in various complexes to address its active site architecture. Unexpectedly, the quaternary structure of WbkC was shown to be different from that previously observed for other sugar N-formyltransferases. Additionally, the structures revealed a second binding site for a GDP molecule distinct from that required for GDP-perosamine positioning. In keeping with this additional binding site, kinetic data with the wild type enzyme revealed complex patterns. Removal of GDP binding by mutating Phe 142 to an alanine residue resulted in an enzyme variant displaying normal Michaelis-Menten kinetics. These data suggest that this nucleotide binding pocket plays a role in enzyme regulation. Finally, by using an alternative substrate, we demonstrate that WbkC can be utilized to produce a trideoxysugar not found in nature.

Structural Studies on a Glucosamine/Glucosaminide N-Acetyltransferase.

Glucosamine/glucosaminide N-acetyltransferase or GlmA catalyzes the transfer of an acetyl group from acetyl CoA to the primary amino group of glucosamine. The enzyme from Clostridium acetobutylicum is thought to be involved in cell wall rescue. In addition to glucosamine, GlmA has been shown to function on di- and trisaccharides of glucosamine as well. Here we present a structural and kinetic analysis of the enzyme. For this investigation, eight structures were determined to resolutions of 2.0 Å or better. The overall three-dimensional fold of GlmA places it into the tandem GNAT superfamily. Each subunit of the dimer folds into two distinct domains which exhibit high three-dimensional structural similarity. Whereas both domains bind acetyl CoA, it is the C-terminal domain that is catalytically competent. On the basis of the various structures determined in this investigation, two amino acid residues were targeted for further study: Asp 287 and Tyr 297. Although their positions in the active site suggested that they may play key roles in catalysis by functioning as active site bases and acids, respectively, this was not borne out by characterization of the D287N and Y297F variants. The kinetic properties revealed that both residues were important for substrate binding but had no critical roles as acid/base catalysts. Kinetic analyses also indicated that GlmA follows an ordered mechanism with acetyl CoA binding first followed by glucosamine. The product N-acetylglucosamine is then released prior to CoA. The investigation described herein provides significantly new information on enzymes belonging to the tandem GNAT superfamily.

Examining Reaction Specificity in PvcB, a Source of Diversity in Isonitrile-Containing Natural Products.

Many bacteria produce isonitrile-containing natural products that are derived from aromatic amino acids. The synthetic clusters that control biosynthesis most commonly encode two enzymes, designated PvcA and PvcB, as well as additional enzymes that direct synthesis of the natural product. The PvcA enzyme installs the isonitrile moiety at the amino group of either tyrosine or tryptophan, as dictated by the particular pathway. The common pathway intermediate produced by PvcA is directed toward different ultimate products by PvcB, a member of the family of Fe(2+), α-ketoglutarate-dependent oxygenases. To continue our investigation of the structural and functional properties of the isonitrile biosynthetic pathways, we present here a study of the PvcB homologues from three organisms. Two pathways, derived from Pseudomonas aeruginosa and Xenorhabdus nematophila, produce known products. A third PvcB homologue from Erwinia amylovora is part of an uncharacterized pathway. Our results demonstrate the diversity of reactions catalyzed. Although all PvcB enzymes catalyze the hydroxylation of the tyrosine isonitrile substrate, the elimination of the hydroxyl in Pseudomonas and Erwinia is driven by deprotonation at Cα, resulting in the initial production of an unsaturated tyrosine isonitrile product that then cyclizes to a coumarin derivative. PvcB from Xenorhabdus, in contrast, catalyzes the same oxygenation, but loss of the hydroxyl group is accompanied by decarboxylation of the intermediate. Steady-state kinetic analysis of the three reactions and a docking model for the binding of the tyrosine isonitrile substrate in the PvcB active site highlight subtle differences between the PvcB homologues.

A conserved motif mediates both multimer formation and allosteric activation of phosphoglycerate mutase 5.

Phosphoglycerate mutase 5 (PGAM5) is an atypical mitochondrial Ser/Thr phosphatase that modulates mitochondrial dynamics and participates in both apoptotic and necrotic cell death. The mechanisms that regulate the phosphatase activity of PGAM5 are poorly understood. The C-terminal phosphoglycerate mutase domain of PGAM5 shares homology with the catalytic domains found in other members of the phosphoglycerate mutase family, including a conserved histidine that is absolutely required for catalytic activity. However, this conserved domain is not sufficient for maximal phosphatase activity. We have identified a highly conserved amino acid motif, WDXNWD, located within the unique N-terminal region, which is required for assembly of PGAM5 into large multimeric complexes. Alanine substitutions within the WDXNWD motif abolish the formation of multimeric complexes and markedly reduce phosphatase activity of PGAM5. A peptide containing the WDXNWD motif dissociates the multimeric complex and reduces but does not fully abolish phosphatase activity. Addition of the WDXNWD-containing peptide in trans to a mutant PGAM5 protein lacking the WDXNWD motif markedly increases phosphatase activity of the mutant protein. Our results are consistent with an intermolecular allosteric regulation mechanism for the phosphatase activity of PGAM5, in which the assembly of PGAM5 into multimeric complexes, mediated by the WDXNWD motif, results in maximal activation of phosphatase activity. Our results suggest the possibility of identifying small molecules that function as allosteric regulators of the phosphatase activity of PGAM5.

Evolutionary history and metabolic insights of ancient mammalian uricases.

Uricase is an enzyme involved in purine catabolism and is found in all three domains of life. Curiously, uricase is not functional in some organisms despite its role in converting highly insoluble uric acid into 5-hydroxyisourate. Of particular interest is the observation that apes, including humans, cannot oxidize uric acid, and it appears that multiple, independent evolutionary events led to the silencing or pseudogenization of the uricase gene in ancestral apes. Various arguments have been made to suggest why natural selection would allow the accumulation of uric acid despite the physiological consequences of crystallized monosodium urate acutely causing liver/kidney damage or chronically causing gout. We have applied evolutionary models to understand the history of primate uricases by resurrecting ancestral mammalian intermediates before the pseudogenization events of this gene family. Resurrected proteins reveal that ancestral uricases have steadily decreased in activity since the last common ancestor of mammals gave rise to descendent primate lineages. We were also able to determine the 3D distribution of amino acid replacements as they accumulated during evolutionary history by crystallizing a mammalian uricase protein. Further, ancient and modern uricases were stably transfected into HepG2 liver cells to test one hypothesis that uricase pseudogenization allowed ancient frugivorous apes to rapidly convert fructose into fat. Finally, pharmacokinetics of an ancient uricase injected in rodents suggest that our integrated approach provides the foundation for an evolutionarily-engineered enzyme capable of treating gout and preventing tumor lysis syndrome in human patients.

Structural and functional characterization of Pseudomonas aeruginosa AlgX: role of AlgX in alginate acetylation.

The exopolysaccharide alginate, produced by mucoid Pseudomonas aeruginosa in the lungs of cystic fibrosis patients, undergoes two different chemical modifications as it is synthesized that alter the properties of the polymer and hence the biofilm. One modification, acetylation, causes the cells in the biofilm to adhere better to lung epithelium, form microcolonies, and resist the effects of the host immune system and/or antibiotics. Alginate biosynthesis requires 12 proteins encoded by the algD operon, including AlgX, and although this protein is essential for polymer production, its exact role is unknown. In this study, we present the X-ray crystal structure of AlgX at 2.15 Å resolution. The structure reveals that AlgX is a two-domain protein, with an N-terminal domain with structural homology to members of the SGNH hydrolase superfamily and a C-terminal carbohydrate-binding module. A number of residues in the carbohydrate-binding module form a substrate recognition "pinch point" that we propose aids in alginate binding and orientation. Although the topology of the N-terminal domain deviates from canonical SGNH hydrolases, the residues that constitute the Ser-His-Asp catalytic triad characteristic of this family are structurally conserved. In vivo studies reveal that site-specific mutation of these residues results in non-acetylated alginate. This catalytic triad is also required for acetylesterase activity in vitro. Our data suggest that not only does AlgX protect the polymer as it passages through the periplasm but that it also plays a role in alginate acetylation. Our results provide the first structural insight for a wide group of closely related bacterial polysaccharide acetyltransferases.

Functional characterization of AlgL, an alginate lyase from Pseudomonas aeruginosa.

Alginate lyase (AlgL) catalyzes the cleavage of the polysaccharide alginate through a ß-elimination reaction. In Pseudomonas aeruginosa, algL is part of the alginate biosynthetic operon, and although it is required for alginate biosynthesis, it is not clear why. Steady-state kinetic studies were performed to characterize its substrate specificity and revealed that AlgL operates preferentially on nonacetylated alginate or its precursor mannuronan. Mature alginate is secreted as a partially acetylated polysaccharide, so this observation is consistent with suggestions that AlgL serves to degrade mislocalized alginate that is trapped in the periplasmic space. The k(cat)/K(m) for the reaction increased linearly with the number of residues in the substrate, from 2.1 × 10(5) M(-1) s(-1) for the substrate containing 16 residues to 7.9 × 10(6) M(-1) s(-1) for the substrate with 280 residues. Over the same substrate size range, k(cat) varied between 10 and 30 s(-1). The variation in k(cat)/K(m) with substrate length suggests that AlgL operates in a processive manner. AlgL displayed a surprising lack of stereospecificity, in that it was able to catalyze cleavage adjacent to either mannuronate or guluronate residues in alginate. Thus, the enzyme is able to remove the C5 proton from both mannuronate and guluronate, which are C5 epimers. Exhaustive digestion of alginate by AlgL generated dimeric and trimeric products, which were characterized by (1)H nuclear magnetic resonance spectroscopy and mass spectrometry. Rapid-mixing chemical quench studies revealed that there was no lag in dimer or trimer production, indicating that AlgL operates as an exopolysaccharide lyase.

The enzymes of bacterial census and censorship.

N-Acyl-L-homoserine lactones (AHLs) are a major class of quorum-sensing signals used by Gram-negative bacteria to regulate gene expression in a population-dependent manner, thereby enabling group behavior. Enzymes capable of generating and catabolizing AHL signals are of significant interest for the study of microbial ecology and quorum-sensing pathways, for understanding the systems that bacteria have evolved to interact with small-molecule signals, and for their possible use in therapeutic and industrial applications. The recent structural and functional studies reviewed here provide a detailed insight into the chemistry and enzymology of bacterial communication.

Activity-based metagenomic screening and biochemical characterization of bovine ruminal protozoan glycoside hydrolases.

The rumen, the foregut of herbivorous ruminant animals such as cattle, functions as a bioreactor to process complex plant material. Among the numerous and diverse microbes involved in ruminal digestion are the ruminal protozoans, which are single-celled, ciliated eukaryotic organisms. An activity-based screen was executed to identify genes encoding fibrolytic enzymes present in the metatranscriptome of a bovine ruminal protozoan-enriched cDNA expression library. Of the four novel genes identified, two were characterized in biochemical assays. Our results provide evidence for the effective use of functional metagenomics to retrieve novel enzymes from microbial populations that cannot be maintained in axenic cultures.

Kinetic consequences of replacing the internucleotide phosphorus atoms in DNA with arsenic.

It was claimed in a recent publication that a strain of Halomonadacea bacteria (GFAJ-1) isolated from the arsenic-rich waters of Mono Lake, California is able to substitute arsenic for phosphorus in its macromolecules and small molecule metabolites. In this short Perspective, we consider chemical and biochemical issues surrounding the central claim that Halomonadacea GFAJ-1 is able to survive while incorporating kinetically labile arsenodiester linkages into the backbone of its DNA. Chemical precedents suggest that arsenodiester linkages in the putative arsenic-containing DNA of GFAJ-1 would undergo very rapid hydrolytic cleavage in water at 25 °C with an estimated half-life of 0.06 s. In contrast, the phosphodiester linkages of native DNA undergo spontaneous hydrolysis with a half-life of approximately 30,000,000 y at 25 °C. Overcoming such dramatic kinetic instability in its genetic material would present serious challenges to Halomonadacea GFAJ-1.

Expression, purification, crystallization and preliminary X-ray analysis of Pseudomonas aeruginosa AlgX.

AlgX is a periplasmic protein required for the production of the exopolysaccharide alginate in Pseudomonas sp. and Azotobacter vinelandii. AlgX has been overexpressed and purified and diffraction-quality crystals have been grown using iterative seeding and the hanging-drop vapor-diffusion method. The crystals grew as flat plates with unit-cell parameters a = 46.4, b = 120.6, c = 86.9 A, beta = 95.7 degrees . The crystals exhibited the symmetry of space group P2(1) and diffracted to a minimum d-spacing of 2.1 A. On the basis of the Matthews coefficient (V(M) = 2.25 A(3) Da(-1)), two molecules were estimated to be present in the asymmetric unit.

Reactivity and reaction order in acylhomoserine lactone formation by Pseudomonas aeruginosa RhlI.

The formation of N-butyrylhomoserine lactone catalyzed by RhlI has been investigated by transient-state kinetic methods. A single intermediate, assigned to N-butyryl- S-adenosylmethionine, was observed. Under single-turnover conditions, the intermediate formed with a rate constant of 4.0 +/- 0.2 s (-1) and decayed with a rate constant of 3.7 +/- 0.2 s (-1). No other intermediates were detected, demonstrating that the RhlI reaction proceeds via acylation of S-adenosylmethionine, followed by lactonization. S-Adenosylhomocysteine acted as a pseudosubstrate, in that it did not undergo either acylation or lactonization but did induce the deacylation of butyryl-acyl carrier protein. The K m for S-adenosylhomocysteine was approximately 15-fold higher than the K m for S-adenosylmethionine. The reactivities of acylated and unacylated sulfonium ions that were analogues of S-adenosylmethionine were investigated by computational methods. The calculations indicated that acylation of the substrate amino group activated the substrate for lactonization, by allowing the carboxyl group oxygen to approach more closely the methylene carbon to which it adds. This observation provides a satisfying chemical rationale for the order of the individual reactions in the catalytic cycle.

Chemical mechanism and specificity of the C5-mannuronan epimerase reaction.

C5-mannuronan epimerase catalyzes the formation of alpha-L-guluronate residues from beta-D-mannuronate residues in the synthesis of the linear polysaccharide alginate. The reaction requires the abstraction of a proton from C5 of the residue undergoing epimerization followed by re-protonation on the opposite face. Rapid-mixing chemical quench experiments were conducted to determine the nature of the intermediate formed upon proton abstraction in the reaction catalyzed by the enzyme from Pseudomonas aeruginosa. Colorimetric and HPLC analysis of quenched samples indicated that shortened oligosaccharides containing an unsaturated sugar residue form as transient intermediates in the epimerization reaction. This suggests that the carbanion is stabilized by glycal formation, concomitant with cleavage of the glycosidic bond between the residue undergoing epimerization and the adjacent residue. The time dependence of glycal formation suggested that slow steps flank the chemical steps in the catalytic cycle. Solvent isotope effects on V and V/K were unity, consistent with a catalytic cycle in which chemistry is not rate-limiting. The specificity of the epimerase with regard to neighboring residues was examined, and it was determined that the enzyme showed no bias for mannuronate residues adjacent to guluronates versus those adjacent to mannuronates. Proton abstraction and sugar epimerization were irreversible. Existing guluronate residues already present in the polysaccharide were not converted to mannuronates, nor was incorporation of solvent deuterium into existing mannuronates observed.

Pseudomonas aeruginosa C5-mannuronan epimerase: steady-state kinetics and characterization of the product.

Alginate is a major constituent of mature biofilms produced by Pseudomonas aeruginosa. The penultimate step in the biosynthesis of alginate is the conversion of some beta-D-mannuronate residues in the polymeric substrate polymannuronan to alpha-L-guluronate residues in a reaction catalyzed by C5-mannuronan epimerase. Specificity studies conducted with size-fractionated oligomannuronates revealed that the minimal substrate contained nine monosaccharide residues. The maximum velocity of the reaction increased from 0.0018 to 0.0218 s(-1) as the substrate size increased from 10 to 20 residues, and no additional increase in kcat was observed for substrates up to 100 residues in length. The Km decreased from 80 microM for a substrate containing fewer than 15 residues to 4 microM for a substrate containing more than 100 residues. In contrast to C5-mannuronan epimerases that have been characterized in other bacterial species, P. aeruginosa C5-mannuronan epimerase does not require Ca2+ for activity, and the Ca2+-alginate complex is not a substrate for the enzyme. Analysis of the purified, active enzyme by inductively coupled plasma-emission spectroscopy revealed that no metals were present in the protein. The pH dependence of the kinetic parameters revealed that three residues on the enzyme which all have a pKa of approximately 7.6 must be protonated for catalysis to occur. The composition of the polymeric product of the epimerase reaction was analyzed by 1H NMR spectroscopy, which revealed that tracts of adjacent guluronate residues were readily formed. The reaction reached an apparent equilibrium when the guluronate composition of the polymer was 75%.

Update on ureide degradation in legumes.

Warm season N2-fixing legumes move fixed N from the nodules to the aerial portions of the plant primarily in the form of ureides, allantoin and allantoate, oxidation products of purines synthesized de novo in the nodule. Ureides are also products of purine turnover in senescing tissues, such as seedling cotyledons. A combination of biochemical and molecular approaches in both crop and model species has shed new light on the metabolic pathways involved in both the synthesis and degradation of allantoin. Improved understanding of ureide biochemistry includes two 'additional' enzymatic steps in the conversion of uric acid to allantoin in the nodule and the mechanism of allantoin and allantoate breakdown in leaf tissue. Ureide accumulation and metabolism in leaves have also been implicated in the feedback inhibition of N2-fixation under water limitation. Sensitivity to water deficit differs among soybean cultivars. Manganese supplementation has been shown to modify relative susceptibility or tolerance to this process in a cultivar-dependent manner. A discussion of the potential roles for ureides and manganese in the feedback inhibition of N2-fixation under water limitation is presented. The existing data are examined in relation to potential changes in both aerial carbon and nitrogen supply under water deficit.

Oxanosine is a substrate of adenosine deaminase. Implications for the quest for a toxicological marker for nitrosation activity.

Oxanosine 3r, 5-amino-3-beta-(d-ribofuranosyl)-3H-imidazo[4,5-d][1,3]oxazine-7-one, was isolated as a novel nucleoside antibiotic in 1981 from Streptomyces capreolus MG265-CF3. Oxanosine became relevant in toxicology in 1996 with the discovery that it is formed in nitrosative guanosine deamination. As part of studies of the mechanism of oxanosine formation, the synthesis was attempted of [7- 18O]oxanosine by enzymatic 16O/18O-exchange with adenosine deaminase (ADA) in analogy to the synthesis of [6- 18O]guanosine from 2-amino-6-chloropurine. Unexpectedly, it was discovered that the incubation of oxanosine 3r with ADA in sodium phosphate buffer (pH = 7.4) results in 1-beta-(d-ribofuranosyl)-5-ureido-1H-imidazole-4-carboxylic acid 4r. The reaction of the 2'-deoxyribose derivative 3d forms 4d in analogy. The reaction products were separated by preparative RP-HPLC and characterized by LC/MS and MS/MS analyses and UV/vis and NMR spectroscopy, and NMR assignments were corroborated by GIAO and GIAO-PCM calculations. Reaction in H2 18O leads to 18O-incorporation at C7. The hydrolysis of 3 to 4 can be rationalized on the basis of the known mode of action of ADA, and an explanation is provided for ADA's accomplishment of the "usual" substitution at C6 of adenosine (addition to the exocyclic bond) and the "lactone hydrolysis" of oxanosine (addition to the endocyclic double bond). The Michaelis-Menten constant of Km = 1.0 (+/-0.2) mM was measured for oxanosine. Implications are discussed for studies of nitrosative deamination of nucleosides, nucleotides, and oligonucleotides.