Complex structure of Bacillus subtilis RibG: the reduction mechanism during riboflavin biosynthesis

Bacterial RibG is a potent target for antimicrobial agents, because it catalyzes consecutive deamination and reduction steps in the riboflavin biosynthesis. In the N-terminal deaminase domain of Bacillus subtilis RibG, structure-based mutational analyses demonstrated that Glu51 and Lys79 are essential for the deaminase activity. In the C-terminal reductase domain, the complex structure with the substrate at 2.56-A resolution unexpectedly showed a ribitylimino intermediate bound at the active site, and hence suggested that the ribosyl reduction occurs through a Schiff base pathway. Lys151 seems to have evolved to ensure specific recognition of the deaminase product rather than the substrate. Glu290, instead of the previously proposed Asp199, would seem to assist in the proton transfer in the reduction reaction. A detailed comparison reveals that the reductase and the pharmaceutically important enzyme, dihydrofolate reductase involved in the riboflavin and folate biosyntheses, share strong conservation of the core structure, cofactor binding, catalytic mechanism, even the substrate binding architecture.     

The crystal structure of the bifunctional deaminase/reductase RibD of the riboflavin biosynthetic pathway in Escherichia coli: implications for the reductive mechanism

We have determined the crystal structure of the bi-functional deaminase/reductase enzyme from Escherichia coli (EcRibD) that catalyzes two consecutive reactions during riboflavin biosynthesis. The polypeptide chain of EcRibD is folded into two domains where the 3D structure of the N-terminal domain (1-145) is similar to cytosine deaminase and the C-terminal domain (146-367) is similar to dihydrofolate reductase. We showed that EcRibD is dimeric and compared our structure to tetrameric RibG, an ortholog from Bacillus subtilis (BsRibG). We have also determined the structure of EcRibD in two binary complexes with the oxidized cofactor (NADP(+)) and with the substrate analogue ribose-5-phosphate (RP5) and superposed these two in order to mimic the ternary complex. Based on this superposition we propose that the invariant Asp200 initiates the reductive reaction by abstracting a proton from the bound substrate and that the pro-R proton from C4 of the cofactor is transferred to C1 of the substrate. A highly flexible loop is found in the reductase active site (159-173) that appears to control cofactor and substrate binding to the reductase active site and was therefore compared to the corresponding Met20 loop of E. coli dihydrofolate reductase (EcDHFR). Lys152, identified by comparing substrate analogue (RP5) coordination in the reductase active site of EcRibD with the homologous reductase from Methanocaldococcus jannaschii (MjaRED), is invariant among bacterial RibD enzymes and could contribute to the various pathways taken during riboflavin biosynthesis in bacteria and yeast.     

Biosynthesis of riboflavin: characterization of the bifunctional deaminase-reductase of Escherichia coli and Bacillus subtilis.

The ribG gene at the 5' end of the riboflavin operon of Bacillus subtilis and a reading frame at 442 kb on the Escherichia coli chromosome (subsequently designated ribD) show similarity with deoxycytidylate deaminase and with the RIB7 gene of Saccharomyces cerevisiae. The ribG gene of B. subtilis and the ribD gene of E. coli were expressed in recombinant E. coli strains and were shown to code for bifunctional proteins catalyzing the second and third steps in the biosynthesis of riboflavin, i.e., the deamination of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (deaminase) and the subsequent reduction of the ribosyl side chain (reductase). The recombinant proteins specified by the ribD gene of E. coli and the ribG gene of B. subtilis were purified to homogeneity. NADH as well as NADPH can be used as a cosubstrate for the reductase of both microorganisms under study. Expression of the N-terminal or C-terminal part of the RibG protein yielded proteins with deaminase or reductase activity, respectively; however, the truncated proteins were rather unstable.     

Crystal structure of Bacillus subtilis guanine deaminase: the first domain-swapped structure in the cytidine deaminase superfamily

Guanine deaminase, a key enzyme in the nucleotide metabolism, catalyzes the hydrolytic deamination of guanine into xanthine. The crystal structure of the 156-residue guanine deaminase from Bacillus subtilis has been solved at 1.17-A resolution. Unexpectedly, the C-terminal segment is swapped to form an intersubunit active site and an intertwined dimer with an extensive interface of 3900 A(2) per monomer. The essential zinc ion is ligated by a water molecule together with His(53), Cys(83), and Cys(86). A transition state analog was modeled into the active site cavity based on the tightly bound imidazole and water molecules, allowing identification of the conserved deamination mechanism and specific substrate recognition by Asp(114) and Tyr(156'). The closed conformation also reveals that substrate binding seals the active site entrance, which is controlled by the C-terminal tail. Therefore, the domain swapping has not only facilitated the dimerization but has also ensured specific substrate recognition. Finally, a detailed structural comparison of the cytidine deaminase superfamily illustrates the functional versatility of the divergent active sites found in the guanine, cytosine, and cytidine deaminases and suggests putative specific substrate-interacting residues for other members such as dCMP deaminases.     

Crystal structure of yeast cytosine deaminase. Insights into enzyme mechanism and evolution

Yeast cytosine deaminase is an attractive candidate for anticancer gene therapy because it catalyzes the deamination of the prodrug 5-fluorocytosine to form 5-fluorouracil. We report here the crystal structure of the enzyme in complex with the inhibitor 2-hydroxypyrimidine at 1.6-A resolution. The protein forms a tightly packed dimer with an extensive interface of 1450 A2 per monomer. The inhibitor was converted into a hydrated adduct as a transition-state analog. The essential zinc ion is ligated by the 4-hydroxyl group of the inhibitor together with His62, Cys91, and Cys94 from the protein. The enzyme shares similar active-site architecture to cytidine deaminases and an unusually high structural homology to 5-aminoimidazole-4-carboxamide-ribonucleotide transformylase and thereby may define a new superfamily. The unique C-terminal tail is involved in substrate specificity and also functions as a gate controlling access to the active site. The complex structure reveals a closed conformation, suggesting that substrate binding seals the active-site entrance so that the catalytic groups are sequestered from solvent. A comparison of the crystal structures of the bacterial and fungal cytosine deaminases provides an elegant example of convergent evolution, where starting from unrelated ancestral proteins, the same metal-assisted deamination is achieved through opposite chiral intermediates within distinctly different active sites.     

Sub-families of alpha/beta barrel enzymes: a new adenine deaminase family

No gene coding for an adenine deaminase has been described in eukaryotes. However, physiological and genetical evidence indicates that adenine deaminases are present in the ascomycetes. We have cloned and characterised the genes coding for the adenine deaminases of Aspergillus nidulans, Saccharomyces cerevisiae and Schizosaccharomyces pombe. The A.nidulans gene was expressed in Escherichia coli and the purified enzyme shows adenine but not adenosine deaminase activity. The open reading frames coded by the three genes are very similar and obviously related to the bacterial and eukaryotic adenosine deaminases rather than to the bacterial adenine deaminases. The latter are related to allantoinases, ureases and dihydroorotases. The fungal adenine deaminases and the homologous adenosine deaminases differ in a number of residues, some of these being clearly involved in substrate specificity. Other prokaryotic enzymes in the database, while clearly related to the above, do not fit into either sub-class, and may even have a different specificity. These results imply that adenine deaminases have appeared twice in the course of evolution, from different ancestral enzymes constructed both around the alpha/beta barrel scaffold.     

Crystal structure of NAD(P)H:flavin oxidoreductase from Escherichia coli.

Flavin reductases use flavins as substrates and are distinct from flavoenzymes which have tightly bound flavins. The reduced flavin can serve to reduce ferric complexes and iron proteins. In Escherichia coli, reactivation of ribonucleotide reductase is achieved by reduced flavins produced by flavin reductase. The crystal structure of E. coli flavin reductase reveals that the enzyme structure is similar to the structures of the ferredoxin reductase family of flavoproteins despite very low sequence similarities. The main difference between flavin reductase and structurally related flavoproteins is that there is no binding site for the AMP moiety of FAD. The direction of the helix in the flavin binding domain, corresponding to the phosphate binding helix in the flavoproteins, is also slightly different and less suitable for phosphate binding. Interactions for flavin substrates are instead provided by a hydrophobic isoalloxazine binding site that also contains a serine and a threonine, which form hydrogen bonds to the isoalloxazine of bound riboflavin in a substrate complex.     

Structures of dCTP deaminase from Escherichia coli with bound substrate and product: reaction mechanism and determinants of mono- and bifunctionality for a family of enzymes

dCTP deaminase (EC 3.5.4.13) catalyzes the deamination of dCTP forming dUTP that via dUTPase is the main pathway providing substrate for thymidylate synthase in Escherichia coli and Salmonella typhimurium. dCTP deaminase is unique among nucleoside and nucleotide deaminases as it functions without aid from a catalytic metal ion that facilitates preparation of a water molecule for nucleophilic attack on the substrate. Two active site amino acid residues, Arg(115) and Glu(138), were identified by mutational analysis as important for activity in E. coli dCTP deaminase. None of the mutant enzymes R115A, E138A, or E138Q had any detectable activity but circular dichroism spectra for all mutant enzymes were similar to wild type suggesting that the overall structure was not changed. The crystal structures of wild-type E. coli dCTP deaminase and the E138A mutant enzyme have been determined in complex with dUTP and Mg(2+), and the mutant enzyme also with the substrate dCTP and Mg(2+). The enzyme is a third member of the family of the structurally related trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii. However, the C-terminal fold is completely different from dUTPases resulting in an active site built from residues from two of the trimer subunits, and not from three subunits as in dUTPases. The nucleotides are well defined as well as Mg(2+) that is tridentately coordinated to the nucleotide phosphate chains. We suggest a catalytic mechanism for the dCTP deaminase and identify structural differences to dUTPases that prevent hydrolysis of the dCTP triphosphate.     

The crystal structure of maleylacetate reductase from Rhizobium sp. strain MTP‐10005 provides insights into the reaction mechanism of enzymes in its original family

Maleylacetate reductase plays a crucial role in catabolism of resorcinol by catalyzing the NAD(P)H‐dependent reduction of maleylacetate, at a carbon–carbon double bond, to 3‐oxoadipate. The crystal structure of maleylacetate reductase from Rhizobium sp. strain MTP‐10005, GraC, has been elucidated by the X‐ray diffraction method at 1.5 Å resolution. GraC is a homodimer, and each subunit consists of two domains: an N‐terminal NADH‐binding domain adopting an α/β structure and a C‐terminal functional domain adopting an α‐helical structure. Such structural features show similarity to those of the two existing families of enzymes in dehydroquinate synthase‐like superfamily. However, GraC is distinct in dimer formation and activity expression mechanism from the families of enzymes. Two subunits in GraC have different structures from each other in the present crystal. One subunit has several ligands mimicking NADH and the substrate in the cleft and adopts a closed domain arrangement. In contrast, the other subunit does not contain any ligand causing structural changes and adopts an open domain arrangement. The structure of GraC reveals those of maleylacetate reductase both in the coenzyme, substrate‐binding state and in the ligand‐free state. The comparison of both subunit structures reveals a conformational change of the Tyr326 loop for interaction with His243 on ligand binding. Structures of related enzymes suggest that His243 is likely a catalytic residue of GraC. Mutational analyses of His243 and Tyr326 support the catalytic roles proposed from structural information. The crystal structure of GraC characterizes the maleylacetate reductase family as a third family in the dehydroquinate synthase‐like superfamily. Proteins 2016; 84:1029–1042. © 2016 Wiley Periodicals, Inc.     

The crystal structure of spermidine synthase with a multisubstrate adduct inhibitor.

Polyamines are essential in all branches of life. Spermidine synthase (putrescine aminopropyltransferase, PAPT) catalyzes the biosynthesis of spermidine, a ubiquitous polyamine. The crystal structure of the PAPT from Thermotoga maritima (TmPAPT) has been solved to 1.5 A resolution in the presence and absence of AdoDATO (S-adenosyl-1,8-diamino-3-thiooctane), a compound containing both substrate and product moieties. This, the first structure of an aminopropyltransferase, reveals deep cavities for binding substrate and cofactor, and a loop that envelops the active site. The AdoDATO binding site is lined with residues conserved in PAPT enzymes from bacteria to humans, suggesting a universal catalytic mechanism. Other conserved residues act sterically to provide a structural basis for polyamine specificity. The enzyme is tetrameric; each monomer consists of a C-terminal domain with a Rossmann-like fold and an N-terminal beta-stranded domain. The tetramer is assembled using a novel barrel-type oligomerization motif.     

Structure and kinetics of a monomeric glucosamine 6-phosphate deaminase: missing link of the NagB superfamily?

Glucosamine 6-phosphate is converted to fructose 6-phosphate and ammonia by the action of the enzyme glucosamine 6-phosphate deaminase, NagB. This reaction is the final step in the specific GlcNAc utilization pathway and thus decides the metabolic fate of GlcNAc. Sequence analyses suggest that the NagB "superfamily" consists of three main clusters: multimeric and allosterically regulated glucosamine-6-phosphate deaminases (exemplified by Escherichia coli NagB), phosphogluconolactonases, and monomeric hexosamine-6-phosphate deaminases. Here we present the three-dimensional structure and kinetics of the first member of this latter group, the glucosamine-6-phosphate deaminase, NagB, from Bacillus subtilis. The structures were determined in ligand-complexed forms at resolutions around 1.4 Angstroms. BsuNagB is monomeric in solution and as a consequence is active (k(cat) 28 s(-1), K(m(app)) 0.13 mM) without the need for allosteric activators. A decrease in activity at high substrate concentrations may reflect substrate inhibition (with K(i) of approximately 4 mM). The structure completes the NagB superfamily structural landscape and thus allows further interrogation of genomic data in terms of the regulation of NagB and the metabolic fate(s) of glucosamine 6-phosphate.     

Kinetic modeling of riboflavin biosynthesis in Bacillus subtilis under production conditions

To study the network dynamics of the riboflavin biosynthesis pathway and to identify potential bottlenecks in the system, an ordinary differential equation-based model was constructed using available literature data for production strains. The results confirmed that the RibA protein is rate limiting in the pathway. Under the conditions investigated, we determined a potential limiting order of the remaining enzymes under increased RibA concentration (>0.102 mM) and therefore higher riboflavin production (>0.045 mmol g _CDW ^?1 h^?1 and 0.0035 mM s^?1, respectively). The reductase activity of RibG and lumazine synthase (RibH) might be the next most limiting steps. The computational minimization of the enzyme concentrations of the pathway suggested the need for a greater RibH concentration (0.251 mM) compared with the other enzymes (RibG: 0.188 mM, RibB: 0.023 mM).     

Cloning and sequence analysis of D-erythrulose reductase from chicken: its close structural relation to tetrameric carbonyl reductases

Sequence analysis of a cDNA for D-erythrulose reductase from chicken liver showed that the deduced open reading frame encodes the protein with a molecular mass of 26 kDa consisting of 246 amino acids. Although the reductase shares more than 60% identity in the amino acid sequence with the mouse tetrameric carbonyl reductase, these two enzymes have many biochemical differences; their substrate specificity, subcellular localization, organ distribution, etc. A three-dimensional structure of D-erythrulose reductase was predicted by comparative modeling based on the structure of the tetrameric carbonyl reductase (PDB entry = 1CYD). Most of the residues at the active site (within 4 A from the ligand) of the carbonyl reductase were also conserved in the D-erythrulose reductase. Nevertheless, Val190 and Leu146 in the active site of the tetrameric carbonyl reductase were substituted in the D-erythrulose reductase by Asn192 and His148, respectively. The substitutions in the active sites may be related to the difference in substrate specificity of the two enzymes. The phylogenic analysis of D-erythrulose reductase and the other related proteins suggests that the protein described as a carbonyl reductase D-erythrulose reductase.     

Crystal structures of Salmonella typhimurium biodegradative threonine deaminase and its complex with CMP provide structural insights into ligand-induced oligomerization and enzyme activation

Two different pyridoxal 5'-phosphate-containing l-threonine deaminases (EC 4.3.1.19), biosynthetic and biodegradative, which catalyze the deamination of l-threonine to alpha-ketobutyrate, are present in Escherichia coli and Salmonella typhimurium. Biodegradative threonine deaminase (TdcB) catalyzes the first reaction in the anaerobic breakdown of l-threonine to propionate. TdcB, unlike the biosynthetic threonine deaminase, is insensitive to l-isoleucine and is activated by AMP. In the present study, TdcB from S. typhimurium was cloned and overexpressed in E. coli. In the presence of AMP or CMP, the recombinant enzyme was converted to the tetrameric form accompanied by significant enzyme activation. To provide insights into ligand-mediated oligomerization and enzyme activation, crystal structures of S. typhimurium TdcB and its complex with CMP were determined. In the native structure, TdcB is in a dimeric form, whereas in the TdcB.CMP complex, it exists in a tetrameric form with 222 symmetry and appears as a dimer of dimers. Tetrameric TdcB binds to four molecules of CMP, two at each of the dimer interfaces. Comparison of the dimer structure in the ligand (CMP)-free and -bound forms suggests that the changes induced by ligand binding at the dimer interface are essential for tetramerization. The differences observed in the tertiary and quaternary structures of TdcB in the absence and presence of CMP appear to account for enzyme activation and increased binding affinity for l-threonine. Comparison of TdcB with related pyridoxal 5'-phosphate-dependent enzymes points to structural and mechanistic similarities.     

Tetrameric dipeptidyl peptidase I directs substrate specificity by use of the residual pro-part domain

The crystal structure of mature dipeptidyl peptidase I reveals insight into the unique tetrameric structure, substrate binding and activation of this atypical papain family peptidase. Each subunit is composed of three peptides. The heavy and light chains form the catalytic domain, which adopts the papain fold. The residual pro-part forms a beta-barrel with the carboxylate group of Asp1 pointing towards the substrate amino-terminus. The tetrameric structure appears to stabilize the association of the two domains and encloses a 12700 A3 spherical cavity. The tetramer contains six chloride ions, one buried in each S2 pocket and two at subunit interfaces.     

A single zinc ion is sufficient for an active Trypanosoma brucei tRNA editing deaminase

Editing of adenosine (A) to inosine (I) at the first anticodon position in tRNA is catalyzed by adenosine deaminases acting on tRNA (ADATs). This essential reaction in bacteria and eukarya permits a single tRNA to decode multiple codons. Bacterial ADATa is a homodimer with two bound essential Zn(2+). The ADATa crystal structure revealed residues important for substrate binding and catalysis; however, such high resolution structural information is not available for eukaryotic tRNA deaminases. Despite significant sequence similarity among deaminases, we continue to uncover unexpected functional differences between Trypanosoma brucei ADAT2/3 (TbADAT2/3) and its bacterial counterpart. Previously, we demonstrated that TbADAT2/3 is unique in catalyzing two different deamination reactions. Here we show by kinetic analyses and inductively coupled plasma emission spectrometry that wild type TbADAT2/3 coordinates two Zn(2+) per heterodimer, but unlike any other tRNA deaminase, mutation of one of the key Zn(2+)-coordinating cysteines in TbADAT2 yields a functional enzyme with a single-bound zinc. These data suggest that, at least, TbADAT3 may play a role in catalysis via direct coordination of the catalytic Zn(2+). These observations raise the possibility of an unusual Zn(2+) coordination interface with important implications for the function and evolution of editing deaminases.     

Deduced amino acid sequence of Escherichia coli adenosine deaminase reveals evolutionarily conserved amino acid residues: implications for catalytic function

The goal of the research reported here is to identify evolutionarily conserved amino acid residues associated with enzymatic deamination of adenosine. To do this, we isolated molecular clones of the Escherichia coli adenosine deaminase gene by functional complementation of adenosine deaminase deficient bacteria and deduced the amino acid sequence of the enzyme from the nucleotide sequence of the gene. Nucleotide sequence analysis revealed the presence of a 996-nucleotide open reading frame encoding a protein of 332 amino acids having a molecular weight of 36,345. The deduced amino acid sequence of the E. coli enzyme has approximately 33% identity with those of the mammalian adenosine deaminases. With conservative amino acid substitutions the overall sequence homology approaches 50%, suggesting that the structures and functions of the mammalian and bacterial enzymes are similar. Additional amino acid sequence analysis revealed specific residues that are conserved among all three adenosine deaminases and four AMP deaminases for which sequence information is currently available. In view of previously published enzymological data and the conserved amino acid residues identified in this study, we propose a model to account for the enzyme-catalyzed hydrolytic deamination of adenosine. Potential catalytic roles are assigned to the conserved His 214, Cys 262, Asp 295, and Asp 296 residues of mammalian adenosine deaminases and the corresponding conserved amino acid residues in bacterial adenosine deaminase and the eukaryotic AMP deaminases.     

Alanine-scanning mutagenesis reveals a cytosine deaminase mutant with altered substrate preference

Suicide gene therapy of cancer is a method whereby cancerous tumors can be selectively eradicated while sparing damage to normal tissue. This is accomplished by delivering a gene, encoding an enzyme capable of specifically converting a nontoxic prodrug into a cytotoxin, to cancer cells followed by prodrug administration. The Escherichia coli gene, codA, encodes cytosine deaminase and is introduced into cancer cells followed by administration of the prodrug 5-fluorocytosine (5-FC). Cytosine deaminase converts 5-FC into cytotoxic 5-fluorouracil, which leads to tumor-cell eradication. One limitation of this enzyme/prodrug combination is that 5-FC is a poor substrate for bacterial cytosine deaminase. The crystal structure of bacterial cytosine deaminase (bCD) reveals that a loop structure in the active site pocket of wild-type bCD comprising residues 310-320 undergoes a conformational change upon cytosine binding, making several contacts to the pyrimidine ring. Alanine-scanning mutagenesis was used to investigate the structure-function relationship of amino acid residues within this region, especially with regard to substrate specificity. Using an E. coli genetic complementation system, seven active mutants were identified (F310A, G311A, H312A, D314A, V315A, F316A, and P318A). Further characterization of these mutants reveals that mutant F316A is 14-fold more efficient than the wild-type at deaminating cytosine to uracil. The mutant D314A enzyme demonstrates a dramatic decrease in cytosine activity (17-fold) as well as a slight increase in activity toward 5-FC (2-fold), indicating that mutant D314A prefers the prodrug over cytosine by almost 20-fold, suggesting that it may be a superior suicide gene.     

Gene cloning and characterization of a deaminase from the 4-amino-3-hydroxybenzoate-assimilating Bordetella sp. strain 10d

The 4-amino-3-hydroxybenzoate-assimilating Bordetella sp. strain 10d produces a deaminase that catalyzes the deamination of 2-amino-5-carboxymuconic 6-semialdehyde. A gene encoding the deaminase, ahdB , was cloned and expressed in Escherichia coli; ahdB is located downstream from the previously reported genes encoding 4-amino-3-hydroxybenzoate 2,3-dioxygenase ( ahdA ) and a LysR-type regulator. The deduced amino acid sequence of ahdB shows 30–33% identity to those of previously reported 2-aminomuconate deaminases. We identified a region (RAGDFLXVSG) conserved in AhdB and three other deaminases. The recombinant enzyme AhdB was purified to homogeneity. After a coupled enzyme assay with purified AhdA, AhdB, and the substrate 4-amino-3-hydroxybenzoate, the final product, formed by the action of AhdA, AhdB, and by nonenzymatic decarboxylation, was identified by HPLC, MS, and ¹H-nuclear magnetic resonance analyses as 2-hydroxymuconic 6-semialdehyde.