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1.  Active-site models for complexes of quinolinate synthase with substrates and intermediates 
Structural studies of quinolinate synthase suggest a model for the enzyme–substrate complex and an enzyme–intermediate complex with a [4Fe–4S] cluster.
Quinolinate synthase (QS) catalyzes the condensation of iminoaspartate and dihydroxyacetone phosphate to form quinolinate, the universal precursor for the de novo biosynthesis of nicotinamide adenine dinucleotide. QS has been difficult to characterize owing either to instability or lack of activity when it is overexpressed and purified. Here, the structure of QS from Pyrococcus furiosus has been determined at 2.8 Å resolution. The structure is a homodimer consisting of three domains per protomer. Each domain shows the same topology with a four-stranded parallel β-sheet flanked by four α-helices, suggesting that the domains are the result of gene triplication. Biochemical studies of QS indicate that the enzyme requires a [4Fe–4S] cluster, which is lacking in this crystal structure, for full activity. The organization of domains in the protomer is distinctly different from that of a monomeric structure of QS from P. horikoshii [Sakuraba et al. (2005 ▶), J. Biol. Chem. 280, 26645–26648]. The domain arrangement in P. furiosus QS may be related to protection of cysteine side chains, which are required to chelate the [4Fe–4S] cluster, prior to cluster assembly.
PMCID: PMC3760129  PMID: 23999292
NAD biosynthesis; iron–sulfur cluster; gene triplication
2.  Structural basis of the substrate specificity of Bacillus cereus adenosine phosphorylase 
Adenosine phosphorylase from B. cereus shows a strong preference for adenosine over other 6-oxopurine nucleosides. Mutation of Asp204 to asparagine reduces the efficiency of adenosine cleavage but does not affect inosine cleavage, effectively reversing the substrate specificity. The structures of D204N complexes explain these observations.
Purine nucleoside phosphorylases catalyze the phosphorolytic cleavage of the glycosidic bond of purine (2′-deoxy)nucleosides, generating the corresponding free base and (2′-deoxy)­ribose 1-phosphate. Two classes of PNPs have been identified: homotrimers specific for 6-oxopurines and homohexamers that accept both 6-oxopurines and 6-aminopurines. Bacillus cereus adenosine phosphorylase (AdoP) is a hexameric PNP; however, it is highly specific for 6-aminopurines. To investigate the structural basis for the unique substrate specificity of AdoP, the active-site mutant D204N was prepared and kinetically characterized and the structures of the wild-type protein and the D204N mutant complexed with adenosine and sulfate or with inosine and sulfate were determined at high resolution (1.2–1.4 Å). AdoP interacts directly with the preferred substrate through a hydrogen-bond donation from the catalytically important residue Asp204 to N7 of the purine base. Comparison with Escherichia coli PNP revealed a more optimal orientation of Asp204 towards N7 of adenosine and a more closed active site. When inosine is bound, two water molecules are interposed between Asp204 and the N7 and O6 atoms of the nucleoside, thus allowing the enzyme to find alternative but less efficient ways to stabilize the transition state. The mutation of Asp204 to asparagine led to a significant decrease in catalytic efficiency for adenosine without affecting the efficiency of inosine cleavage.
PMCID: PMC3282621  PMID: 22349225
purine nucleoside phosphorylases; substrate specificity; reversal of substrate preference; transition-state stabilization
3.  A novel mechanism of sulfur transfer catalyzed by O-acetylhomoserine sulfhydrylase in the methionine-biosynthetic pathway of Wolinella succinogenes  
MetY is the first reported structure of an O-acetylhomoserine sulfhydrylase that utilizes a protein thiocarboxylate intermediate as the sulfur source in a novel methionine-biosynthetic pathway instead of catalyzing a direct sulfhydrylation reaction.
O-Acetylhomoserine sulfhydrylase (OAHS) is a pyridoxal 5′-­phosphate (PLP) dependent sulfide-utilizing enzyme in the l-cysteine and l-methionine biosynthetic pathways of various enteric bacteria and fungi. OAHS catalyzes the conversion of O-acetylhomoserine to homocysteine using sulfide in a process known as direct sulfhydrylation. However, the source of the sulfur has not been identified and no structures of OAHS have been reported in the literature. Here, the crystal structure of Wolinella succinogenes OAHS (MetY) determined at 2.2 Å resolution is reported. MetY crystallized in space group C2 with two monomers in the asymmetric unit. Size-exclusion chromatography, dynamic light scattering and crystal packing indicate that the biological unit is a tetramer in solution. This is further supported by the crystal structure, in which a tetramer is formed using a combination of non­­crystallographic and crystallographic twofold axes. A search for structurally homologous proteins revealed that MetY has the same fold as cystathionine γ-lyase and methionine γ-lyase. The active sites of these enzymes, which are also PLP-dependent, share a high degree of structural similarity, suggesting that MetY belongs to the γ-elimination subclass of the Cys/Met metabolism PLP-dependent family of enzymes. The structure of MetY, together with biochemical data, provides insight into the mechanism of sulfur transfer to a small molecule via a protein thiocarboxylate intermediate.
PMCID: PMC3176619  PMID: 21931214
Wolinella succinogenes; O-acetylhomoserine sulfhydrylases; pyridoxal 5′-phosphate; γ-elimination; direct sulfhydrylation; Cys/Met metabolism; O-acetylhomoserine; protein thiocarboxylate
4.  Structure of trifunctional THI20 from yeast 
This study presents the crystal structure of the trifunctional THI20 from S. cerevisiae, an enzyme with an N-terminal HMP kinase (ThiD-like) domain and a C-terminal thiaminase II (TenA-like) domain. In addition, two distinct structural classes of TenA have been identified by comparison to other known structures.
In a recently characterized thiamin-salvage pathway, thiamin-degradation products are hydrolyzed by thiaminase II, yielding 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP). This compound is an intermediate in thiamin biosynthesis that, once phosphorylated by an HMP kinase, can be used to synthesize thiamin monophosphate. Here, the crystal structure of Saccharomyces cerevisiae THI20, a trifunctional enzyme containing an N-terminal HMP kinase/HMP-P kinase (ThiD-like) domain and a C-terminal thia­min­ase II (TenA-like) domain, is presented. Comparison to structures of the monofunctional enzymes reveals that while the ThiD-like dimer observed in THI20 resembles other ThiD structures, the TenA-like domain, which is tetrameric in all previously reported structures, forms a dimer. Similarly, the active site of the ThiD-like domain of THI20 is highly similar to other known ThiD enzymes, while the TenA-like active site shows unique features compared with previously structurally characterized TenAs. In addition, a survey of known TenA structures revealed two structural classes, both of which have distinct conserved features. The TenA domain of THI20 possesses some features of both classes, consistent with its ability to hydrolyze both thiamin and the thiamin-degradation product 2-methyl-4-amino-5-aminomethylpyrimidine.
PMCID: PMC3169313  PMID: 21904031
thiamin salvage; thiamin metabolism; TenA; ThiD; thiaminases; HMP kinases
5.  Structural and kinetic insights into the mechanism of 5-hydroxyisourate hydrolase from Klebsiella pneumoniae  
The crystal structure of 5-hydroxyisourate hydrolase from K. pneumoniae and the steady-state kinetic parameters of the native enzyme as well as several mutants provide insights into the catalytic mechanism of this enzyme and the possible roles of the active-site residues.
The stereospecific oxidative degradation of uric acid to (S)-­allantoin has recently been demonstrated to proceed via two unstable intermediates and requires three separate enzymatic reactions. The second step of this reaction, the conversion of 5-­hydroxyisourate (HIU) to 2-oxo-4-hydroxy-4-­carboxy-5-ureidoimidazoline, is catalyzed by HIU hydrolase (HIUH). The high-resolution crystal structure of HIUH from the opportunistic pathogen Klebsiella pneumoniae (KpHIUH) has been determined. KpHIUH is a homotetrameric protein that, based on sequence and structural similarity, belongs to the transthyretin-related protein family. In addition, the steady-state kinetic parameters for this enzyme and four active-site mutants have been measured. These data provide valuable insight into the functional roles of the active-site residues. Based upon the structural and kinetic data, a mechanism is proposed for the KpHIUH-catalyzed reaction.
PMCID: PMC3144850  PMID: 21795808
purine catabolism; ureides; uric acid degradation; Hpx genes; transthyretin-like proteins
6.  Structure of grouper iridovirus purine nucleoside phosphorylase 
The crystal structure of purine nucleoside phosphorylase from grouper iridovirus was solved at 2.38 Å resolution.
Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides to the corresponding free bases and ribose 1-phosphate. The crystal structure of grouper iridovirus PNP (givPNP), corresponding to the first PNP gene to be found in a virus, was determined at 2.4 Å resolution. The crystals belonged to space group R3, with unit-cell parameters a = 193.0, c = 105.6 Å, and contained four protomers per asymmetric unit. The overall structure of givPNP shows high similarity to mammalian PNPs, having an α/β structure with a nine-stranded mixed β-­barrel flanked by a total of nine α-helices. The predicted phosphate-binding and ribose-binding sites are occupied by a phosphate ion and a Tris molecule, respectively. The geo­metrical arrangement and hydrogen-bonding patterns of the phosphate-binding site are similar to those found in the human and bovine PNP structures. The enzymatic activity assay of givPNP on various substrates revealed that givPNP can only accept 6-oxopurine nucleosides as substrates, which is also suggested by its amino-acid composition and active-site architecture. All these results suggest that givPNP is a homologue of mammalian PNPs in terms of amino-acid sequence, molecular mass, substrate specificity and overall structure, as well as in the composition of the active site.
PMCID: PMC2815667  PMID: 20124695
purine salvage; phosphorolysis; viral proteins; Tris-binding site
7.  Complexes of Thermotoga maritima S-adenosylmethionine decarboxylase provide insights into substrate specificity 
The crystal structures of activated S-adenosylmethionine decarboxylase (AdoMetDC) from T. maritima and of its complexes with S-adenosylmethionine methyl ester and 5′-deoxy-5′-dimethylthioadenosine have been obtained. Comparison of the structures with that of human AdoMetDC provides insights into the substrate specificity, binding mode, autoprocessing and evolution of the enzyme.
The polyamines putrescine, spermidine and spermine are ubiquitous aliphatic cations and are essential for cellular growth and differentiation. S-Adenosylmethionine decarboxylase (AdoMetDC) is a critical pyruvoyl-dependent enzyme in the polyamine-biosynthetic pathway. The crystal structures of AdoMetDC from humans and plants and of the AdoMetDC proenzyme from Thermotoga maritima have been obtained previously. Here, the crystal structures of activated T. maritima AdoMetDC (TmAdoMetDC) and of its complexes with S-­adenosylmethionine methyl ester and 5′-deoxy-5′-dimethyl­thioadenosine are reported. The results demonstrate for the first time that TmAdoMetDC autoprocesses without the need for additional factors and that the enzyme contains two complete active sites, both of which use residues from both chains of the homodimer. The complexes provide insights into the substrate specificity and ligand binding of AdoMetDC in prokaryotes. The conservation of the ligand-binding mode and the active-site residues between human and T. maritima AdoMetDC provides insight into the evolution of AdoMetDC.
PMCID: PMC2815669  PMID: 20124698
protein evolution; proenzymes; pyruvoyl cofactor; cation–π interactions
8.  Structures of the N47A and E109Q mutant proteins of pyruvoyl-dependent arginine decarboxylase from Methanococcus jannaschii  
The crystal structures of two arginine decarboxylase mutant proteins provide insights into the mechanisms of pyruvoyl-group formation and the decarboxylation reaction.
Pyruvoyl-dependent arginine decarboxylase (PvlArgDC) catalyzes the first step of the polyamine-biosynthetic pathway in plants and some archaebacteria. The pyruvoyl group of PvlArgDC is generated by an internal autoserinolysis reaction at an absolutely conserved serine residue in the proenzyme, resulting in two polypeptide chains. Based on the native structure of PvlArgDC from Methanococcus jannaschii, the conserved residues Asn47 and Glu109 were proposed to be involved in the decarboxylation and autoprocessing reactions. N47A and E109Q mutant proteins were prepared and the three-dimensional structure of each protein was determined at 2.0 Å resolution. The N47A and E109Q mutant proteins showed reduced decarboxylation activity compared with the wild-type PvlArgDC. These residues may also be important for the autoprocessing reaction, which utilizes a mechanism similar to that of the decarboxylation reaction.
PMCID: PMC2467525  PMID: 18391404
arginine decarboxylase; pyruvoyl; decarboxylation; autoprocessing; serinolysis

Results 1-8 (8)