Possible Steps of the Carboxylation of Ribulose-1,5-biphosphate from Intermediates: 2,3-Enediol versus 1,2-Enol.

Ribulose 1,5-bisphosphate (RuBP) undergoes enolization to initiate fixation of atmospheric carbon dioxide in the plant carbon cycle. The known model assumes the binding of RuBP to the Rubisco active site with the subsequent formation of 2,3-enediol (2,3,4-trihydroxypent-2-ene-1,5-diyl diphosphate). In the present study, it is assumed that 1,2-enol (2,3,4-trihydroxypent-1-ene-1,5-diyl diphosphate) can be formed in the enolization step to initiate the carboxylation reaction. We have used Kohn-Sham density functional theory on WB97X-D3/Def2-TZVP levels to compare the reaction barriers in the two ways. We considered the pathways of carboxylation of 1/2-ene (mono/di)ol via the C1 and C2 carbons without taking into account the binding of RuBP to the magnesium ion. Calculations of Gibbs free energies confirm the equal probability of the formation of 2,3-enediol and 1,2-enol. Quantum-chemical modeling of enolization and carboxylation reactions supports the important role of the bridging water molecule and diphosphate groups, which provide proton transfer and lower reaction barriers. The results show that carbon dioxide fixation can occur without a magnesium ion, and binding with C1 can have a lower barrier (~12 kcal/mol) than with C2 (~23 kcal/mol).

Cloning, purification, and characterization of the 6-phosphogluconate dehydrogenase (6 PGDH) from Giardia lamblia.

Giardia lamblia, due to the habitat in which it develops, requires a continuous supply of intermediate compounds that allow it to survive in the host. The pentose phosphate pathway (PPP) provides essential molecules such as NADPH and ribulose-5-phosphate during the oxidative phase of the pathway. One of the key enzymes during this stage is 6-phosphogluconate dehydrogenase (6 PGDH) for generating NADPH. Given the relevance of the enzyme, in the present work, the 6pgdh gene from G. lamblia was amplified and cloned to produce the recombinant protein (Gl-6 PGDH) and characterize it functionally and structurally after the purification of Gl-6 PGDH by affinity chromatography. The results of the characterization showed that the protein has a molecular mass of 54 kDa, with an optimal pH of 7.0 and a temperature of 36-42 °C. The kinetic parameters of Gl-6 PGDH were Km = 49.2 and 139.9 µM (for NADP+ and 6-PG, respectively), Vmax =26.27 µmol*min-1*mg-1, and Kcat = 24.0 s-1. Finally, computational modeling studies were performed to obtain a structural visualization of the Gl-6 PGDH protein. The generation of the model and the characterization assays will allow us to expand our knowledge for future studies of the function of the protein in the metabolism of the parasite.

The structure of a novel membrane-associated 6-phosphogluconate dehydrogenase from Gluconacetobacter diazotrophicus (Gd6PGD) reveals a subfamily of short-chain 6PGDs.

The enzyme 6-phosphogluconate dehydrogenase catalyzes the conversion of 6-phosphogluconate to ribulose-5-phosphate. It represents an important reaction in the oxidative pentose phosphate pathway, producing a ribose precursor essential for nucleotide and nucleic acid synthesis. We succeeded, for the first time, to determine the three-dimensional structure of this enzyme from an acetic acid bacterium, Gluconacetobacter diazotrophicus (Gd6PGD). Active Gd6PGD, a homodimer (70 kDa), was present in both the soluble and the membrane fractions of the nitrogen-fixing microorganism. The Gd6PGD belongs to the newly described subfamily of short-chain (333 AA) 6PGDs, compared to the long-chain subfamily (480 AA; e.g., Ovis aries, Homo sapiens). The shorter amino acid sequence in Gd6PGD induces the exposition of hydrophobic residues in the C-terminal domain. This distinct structural feature is key for the protein to associate with the membrane. Furthermore, in terms of function, the short-chain 6PGD seems to prefer NAD+ over NADP+ , delivering NADH to the membrane-bound NADH dehydrogenase of the microorganisms required by the terminal oxidases to reduce dioxygen to water for energy conservation. ENZYME: ECnonbreakingspace1.1.1.343. DATABASE: Structural data are available in PDB database under the accession number 6VPB.

On a Possible Way to Increase the Efficiency of Photosynthesis.

The paper briefly describes the evolution of the key enzyme of photosynthesis, RuBisCO. Before the emergence of the reaction of carbon dioxide assimilation via photosynthesis, this protein was involved in the methionine metabolism chain. Possibly, for this reason, the carboxylation reaction catalyzed by enzyme proceeds very slowly. In addition to carboxylation, RuBisCO can simultaneously oxidize ribulose bisphosphate, a substrate to which the fixed CO2 is attached. This, in turn, also reduces the effectiveness of photosynthesis. In this regard, the literature discusses various options for increasing plant productivity by creating new forms of RuBisCO or fundamentally different pathways of carbon dioxide assimilation. In this work, we propose a modification of the carboxylation reaction that makes it possible to avoid photorespiration and thus increase the efficiency of photosynthesis.

Ab Initio Molecular Dynamics Simulation and Energetics of the Ribulose-1,5-biphosphate Carboxylation Reaction Catalyzed by Rubisco: Toward Elucidating the Stereospecific Protonation Mechanism.

In the carboxylation reaction catalyzed by ribulose 1,5-bisphosphate (RuBP) carboxylase-oxygenase (Rubisco), which is fundamental to photosynthesis, scission of a C-C bond in the six-carbon gemdiolate intermediate forms a carbanion that must be protonated stereospecifically to form product. It is thought that a conserved lysine side chain (LYS175 in spinach Rubisco), in the immediate vicinity of the carbanion, provides the necessary proton. Here, we endeavor to determine from the electronic-structure calculations whether protonation via this route is energetically possible. The two-dimensional energy surface was mapped to determine the minimum energy path (MEP) using density functional theory (B3LYP) and incorporating basis set superposition and classical (London) dispersion corrections. The potential of mean force (free energy) was then calculated from ab initio molecular dynamics simulations with umbrella sampling in the vicinity of the MEP on the scission-protonation reaction coordinate. MEP calculations were also carried out to evaluate the possibility of an active-site water near the phosphate (P1) of RuBP, with an excess proton positioned at P1, as an alternative facilitator of stereospecific protonation via a classical Grotthuss mechanism. In both cases, the C-C bond scission in the six-carbon intermediate and proton transfer from the donor was found to be concerted and highly asynchronous, without a stable carbanion intermediate. However, the free energy change was unfavorable for direct protonation by the LYS175 side chain. In contrast, the Grotthuss mechanism yielded stable products and an activation energy in good agreement with experiment. It also provides a plausible mechanism for alternative product formed in enzyme mutations at the LYS175 position and is consistent with the observed deuterium isotope effects.

A unique structural domain in Methanococcoides burtonii ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) acts as a small subunit mimic.

The catalytic inefficiencies of the CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) often limit plant productivity. Strategies to engineer more efficient plant Rubiscos have been hampered by evolutionary constraints, prompting interest in Rubisco isoforms from non-photosynthetic organisms. The methanogenic archaeon Methanococcoides burtonii contains a Rubisco isoform that functions to scavenge the ribulose-1,5-bisphosphate (RuBP) by-product of purine/pyrimidine metabolism. The crystal structure of M. burtonii Rubisco (MbR) presented here at 2.6 Å resolution is composed of catalytic large subunits (LSu) assembled into pentamers of dimers, (L2)5, and differs from Rubiscos from higher plants where LSus are glued together by small subunits (SSu) into hexadecameric L8S8 enzymes. MbR contains a unique 29-amino acid insertion near the C terminus, which folds as a separate domain in the structure. This domain, which is visualized for the first time in this study, is located in a similar position to SSus in L8S8 enzymes between LSus of adjacent L2 dimers, where negatively charged residues coordinate around a Mg2+ ion in a fashion that suggests this domain may be important for the assembly process. The Rubisco assembly domain is thus an inbuilt SSu mimic that concentrates L2 dimers. MbR assembly is ligand-stimulated, and we show that only 6-carbon molecules with a particular stereochemistry at the C3 carbon can induce oligomerization. Based on MbR structure, subunit arrangement, sequence, phylogenetic distribution, and function, MbR and a subset of Rubiscos from the Methanosarcinales order are proposed to belong to a new Rubisco subgroup, named form IIIB.

Molecular dynamics studies unravel role of conserved residues responsible for movement of ions into active site of DHBPS.

3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes the conversion of D-ribulose 5-phosphate (Ru5P) to L-3,4-dihydroxy-2-butanone-4-phosphate in the presence of Mg2+. Although crystal structures of DHBPS in complex with Ru5P and non-catalytic metal ions have been reported, structure with Ru5P along with Mg2+ is still elusive. Therefore, mechanistic role played by Mg2+ in the structure of DHBPS is poorly understood. In this study, molecular dynamics simulations of DHBPS-Ru5P complex along with Mg2+ have shown entry of Mg2+ from bulk solvent into active site. Presence of Mg2+ in active site has constrained conformations of Ru5P and has reduced flexibility of loop-2. Formation of hydrogen bonds among Thr-108 and residues - Gly-109, Val-110, Ser-111, and Asp-114 are found to be critical for entry of Mg2+ into active site. Subsequent in silico mutations of residues, Thr-108 and Asp-114 have substantiated the importance of these interactions. Loop-4 of one monomer is being proposed to act as a "lid" covering the active site of other monomer. Further, the conserved nature of residues taking part in the transfer of Mg2+ suggests the same mechanism being present in DHBPS of other microorganisms. Thus, this study provides insights into the functioning of DHBPS that can be used for the designing of inhibitors.

Role of small subunit in mediating assembly of red-type form I Rubisco.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme involved in photosynthetic carbon fixation, converting atmospheric CO2 to organic compounds. Form I Rubisco is a cylindrical complex composed of eight large (RbcL) subunits that are capped by four small subunits (RbcS) at the top and four at the bottom. Form I Rubiscos are phylogenetically divided into green- and red-type. Some red-type enzymes have catalytically superior properties. Thus, understanding their folding and assembly is of considerable biotechnological interest. Folding of the green-type RbcL subunits in cyanobacteria is mediated by the GroEL/ES chaperonin system, and assembly to holoenzyme requires specialized chaperones such as RbcX and RAF1. Here, we show that the red-type RbcL subunits in the proteobacterium Rhodobacter sphaeroides also fold with GroEL/ES. However, assembly proceeds in a chaperone-independent manner. We find that the C-terminal ß-hairpin extension of red-type RbcS, which is absent in green-type RbcS, is critical for efficient assembly. The ß-hairpins of four RbcS subunits form an eight-stranded ß-barrel that protrudes into the central solvent channel of the RbcL core complex. The two ß-barrels stabilize the complex through multiple interactions with the RbcL subunits. A chimeric green-type RbcS carrying the C-terminal ß-hairpin renders the assembly of a cyanobacterial Rubisco independent of RbcX. Our results may facilitate the engineering of crop plants with improved growth properties expressing red-type Rubisco.

Structural modeling and docking studies of ribose 5-phosphate isomerase from Leishmania major and Homo sapiens: a comparative analysis for Leishmaniasis treatment.

Leishmaniases are caused by protozoa of the genus Leishmania and are considered the second-highest cause of death worldwide by parasitic infection. The drugs available for treatment in humans are becoming ineffective mainly due to parasite resistance; therefore, it is extremely important to develop a new chemotherapy against these parasites. A crucial aspect of drug design development is the identification and characterization of novel molecular targets. In this work, through an in silico comparative analysis between the genomes of Leishmania major and Homo sapiens, the enzyme ribose 5-phosphate isomerase (R5PI) was indicated as a promising molecular target. R5PI is an important enzyme that acts in the pentose phosphate pathway and catalyzes the interconversion of d-ribose-5-phosphate (R5P) and d-ribulose-5-phosphate (5RP). R5PI activity is found in two analogous groups of enzymes called RpiA (found in H. sapiens) and RpiB (found in L. major). Here, we present the first report of the three-dimensional (3D) structures and active sites of RpiB from L. major (LmRpiB) and RpiA from H. sapiens (HsRpiA). Three-dimensional models were constructed by applying a hybrid methodology that combines comparative and ab initio modeling techniques, and the active site was characterized based on docking studies of the substrates R5P (furanose and ring-opened forms) and 5RP. Our comparative analyses show that these proteins are structural analogs and that distinct residues participate in the interconversion of R5P and 5RP. We propose two distinct reaction mechanisms for the reversible isomerization of R5P to 5RP, which is catalyzed by LmRpiB and HsRpiA. We expect that the present results will be important in guiding future molecular modeling studies to develop new drugs that are specially designed to inhibit the parasitic form of the enzyme without significant effects on the human analog.

Solvation of CO2 in water: effect of RuBP on CO2 concentration in bundle sheath of C4 plants.

An understanding of the temperature-dependence of solubility of carbon dioxide (CO2) in water is important for many industrial processes. Voluminous work has been done by both quantum chemical methods and molecular dynamics (MD) simulations on the interaction between CO2 and water, but a quantitative evaluation of solubility remains elusive. In this work, we have approached the problem by considering quantum chemically calculated total energies and thermal energies, and incorporating the effects of mixing, hydrogen bonding, and phonon modes. An overall equation relating the calculated free energy and entropy of mixing with the gas-solution equilibrium constant has been derived. This equation has been iteratively solved to obtain the solubility as functions of temperature and dielectric constant. The calculated solubility versus temperature plot excellently matches the observed plot. Solubility has been shown to increase with dielectric constant, for example, by addition of electrolytes. We have also found that at the experimentally reported concentration of enzyme RuBP in bundle sheath cells of chloroplast in C4 green plants, the concentration of CO2 can effectively increase by as much as a factor of 7.1-38.5. This stands in agreement with the observed effective rise in concentration by as much as 10 times.

Structure of Pisum sativum Rubisco with bound ribulose 1,5-bisphosphate.

The first structure of a ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from a pulse crop is reported. Rubisco was purified from Pisum sativum (garden pea) and diffraction-quality crystals were obtained by hanging-drop vapour diffusion in the presence of the substrate ribulose 1,5-bisphosphate. X-ray diffraction data were recorded to 2.20 Šresolution from a single crystal at the Canadian Light Source. The overall quaternary structure of non-activated P. sativum Rubisco highlights the conservation of the form I Rubisco hexadecameric complex. The electron density places the substrate in the active site at the interface of the large-subunit dimers. Lys201 in the active site is not carbamylated as expected for this non-activated structure. Some heterogeneity in the small-subunit sequence is noted, as well as possible variations in the conformation and contacts of ribulose 1,5-bisphosphate in the large-subunit active sites. Overall, the active-site conformation most closely correlates with the `closed' conformation observed in other substrate/inhibitor-bound Rubisco structures.

Quantum chemical modeling of the kinetic isotope effect of the carboxylation step in RuBisCO.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the most important enzyme for the assimilation of carbon into biomass, features a well-known isotope effect with regards to the CO(2) carbon atom. This kinetic isotope effect α = k(12)/k(13) for the carboxylation step of the RuBisCO reaction sequence, and its microscopic origin, was investigated with the help of cluster models and quantum chemical methods [B3LYP/6-31G(d,p)]. We use a recently proposed model for the RuBisCO active site, in which a water molecule remains close to the reaction center during carboxylation of ribulose-1,5-bisphosphate [B. Kannappan, J.E. Gready, J. Am. Chem. Soc. 130 (2008), 15063]. Alternative active-site models and/or computational approaches were also tested. An isotope effect alpha for carboxylation is found, which is reasonably close to the one measured for the overall reaction, and which originates from a simple frequency shift of the bending vibration of (12)CO(2) compared to (13)CO(2). The latter is the dominant mode for the product formation at the transition state.

Structural characterization of a ribose-5-phosphate isomerase B from the pathogenic fungus Coccidioides immitis.

BACKGROUND: Ribose-5-phosphate isomerase is an enzyme that catalyzes the interconversion of ribose-5-phosphate and ribulose-5-phosphate. This family of enzymes naturally occurs in two distinct classes, RpiA and RpiB, which play an important role in the pentose phosphate pathway and nucleotide and co-factor biogenesis. RESULTS: Although RpiB occurs predominantly in bacteria, here we report crystal structures of a putative RpiB from the pathogenic fungus Coccidioides immitis. A 1.9 Å resolution apo structure was solved by combined molecular replacement and single wavelength anomalous dispersion (SAD) phasing using a crystal soaked briefly in a solution containing a high concentration of iodide ions. RpiB from C. immitis contains modest sequence and high structural homology to other known RpiB structures. A 1.8 Å resolution phosphate-bound structure demonstrates phosphate recognition and charge stabilization by a single positively charged residue whereas other members of this family use up to five positively charged residues to contact the phosphate of ribose-5-phosphate. A 1.7 Å resolution structure was obtained in which the catalytic base of C. immitis RpiB, Cys76, appears to form a weakly covalent bond with the central carbon of malonic acid with a bond distance of 2.2 Å. This interaction may mimic that formed by the suicide inhibitor iodoacetic acid with RpiB. CONCLUSION: The C. immitis RpiB contains the same fold and similar features as other members of this class of enzymes such as a highly reactive active site cysteine residue, but utilizes a divergent phosphate recognition strategy and may recognize a different substrate altogether.

Conversion of D-ribulose 5-phosphate to D-xylulose 5-phosphate: new insights from structural and biochemical studies on human RPE.

The pentose phosphate pathway (PPP) confers protection against oxidative stress by supplying NADPH necessary for the regeneration of glutathione, which detoxifies H(2)O(2) into H(2)O and O(2). RPE functions in the PPP, catalyzing the reversible conversion of D-ribulose 5-phosphate to D-xylulose 5-phosphate and is an important enzyme for cellular response against oxidative stress. Here, using structural, biochemical, and functional studies, we show that human D-ribulose 5-phosphate 3-epimerase (hRPE) uses Fe(2+) for catalysis. Structures of the binary complexes of hRPE with D-ribulose 5-phosphate and D-xylulose 5-phosphate provide the first detailed molecular insights into the binding mode of physiological ligands and reveal an octahedrally coordinated Fe(2+) ion buried deep inside the active site. Human RPE folds into a typical (ß/α)(8) triosephosphate isomerase (TIM) barrel with a loop regulating access to the active site. Two aspartic acids are well positioned to carry out the proton transfers in an acid-base type of reaction mechanism. Interestingly, mutating Ser-10 to alanine almost abolished the enzymatic activity, while L12A and M72A mutations resulted in an almost 50% decrease in the activity. The binary complexes of hRPE reported here will aid in the design of small molecules for modulating the activity of the enzyme and altering flux through the PPP.

Putative functional role for the invariant aspartate 263 residue of Rhodospirillum rubrum Rubisco.

Although aspartate residue D263 of Rhodospirillum rubrum Rubisco is close to the active site and invariant in all reported Rubiscos, its possible functional and structural roles in Rubisco activity have not been investigated. We have mutagenised D263 to several selected amino acids (asparagine, alanine, serine, glutamate, and glutamine) to probe possible roles in facilitating proton movements within the active site and maintaining structural positioning of key active-site groups. The mutants have been characterized by kinetic methods and by differential scanning calorimetry (DSC) to examine the effects of the substitutions on the stability of the folded state. We show that D263 is essential for maintaining effective levels of catalysis with the mutations reducing carboxylation variously by up to 100-fold but having less than 10% effect on the carboxylase/oxygenase specificity of the catalytic reaction. Removing the charge of the residue 263 side chain significantly strengthens binding of the activating (carbamylating) CO(2) molecule. In contrast, a charge on the 263 site has only a small influence on binding of the positively charged Mg(2+) ion, suggesting that the local protein structure provides different shielding of the formal charges on the Mg(2+) ion and the epsilon-lysine group of K191. Interestingly, introduction of an internal cavity (D263S and D263A) and insertion of an extra -CH(2)- group (D263E and D263Q) have opposite effects on catalysis, the former relatively small and the latter much larger, suggesting that the extra side-chain group induces a specific structural distortion that inhibits formation of the transition state. As the DSC results show that the mutations only slightly increase the kinetic stability of the folded state, we conclude that the rate-limiting (activated) step of unfolding involves substantial unfolding of the structure but not in the region of site 263. In summary, interaction of D263 with H287 of a largely electrostatic nature appears critical for maintaining correct positioning of catalytic groups in the active site. The conservation of D263 can thus be accounted for by its contribution to the maintenance of a finely tuned structure in this region abutting the active site.

The identification of (3R,4S)-5-fluoro-5-deoxy-D-ribulose-1-phosphate as an intermediate in fluorometabolite biosynthesis in Streptomyces cattleya.

(3R,4S)-5-Fluoro-5-deoxy-D-ribulose-1-phosphate (5-FDRulP) has been identified as the third fluorinated intermediate on the biosynthetic pathway to fluoroacetate and 4-fluorothreonine in Streptomyces cattleya. 5-FDRulP is generated after formation of 5'-fluoro-5'-deoxyadenosine (5'-FDA) and then phosphorolysis of 5'-FDA to 5-fluoro-5-deoxy-D-ribose-1-phosphate (5-FDRP) by the action of a purine nucleoside phosphorylase. An isomerase mediates the conversion of 5-FDRP to 5-FDRulP. The identity of the (3R,4S) diastereoisomer of 5-FDRulP was established by comparative (19)F{(1)H} NMR studies whereby 5-FDRulP that accumulated in a cell free extract of S. cattleya, was treated with a phytase to generate the non-phosphorylated sugar, 5-fluoro-5-deoxy-D-ribulose (5-FDRul). This S. cattleya product was compared to the product of an in-vitro biotransformation where separately 5-fluoro-5-deoxy-D-ribose and 5-fluoro-5-deoxy-D-xylose were converted to 5-fluoro-5-deoxy-D-ribulose and 5-fluoro-5-deoxy-D-xylulose respectively by the action of glucose isomerase. It was demonstrated that 5-fluoro-5-deoxy-D-ribose gave the identical diastereoisomer to that observed from 5-FDRulP.

The physiological role of the ribulose monophosphate pathway in bacteria and archaea.

3-Hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) are the key enzymes of the ribulose monophosphate pathway. This pathway, which was originally found in methylotrophic bacteria, is now recognized as a widespread prokaryotic pathway involved in formaldehyde fixation and detoxification. Recent progress, involving biochemical and genetic approaches in elucidating the physiological functions of HPS and PHI in methylotrophic as well as non-methylotrophic bacteria are described in this review. HPS and PHI orthologs are also found in a variety of archaeal strains. Some archaeal HPS orthologs are fused with other genes to form single ORF (e.g., the hps-phi gene of Pyrococcus spp. and the faeB-hpsB gene of Methanosarcina spp). These fused gene products exhibit functions corresponding to the individual enzyme activities, and are more efficient than equivalent systems made up of discrete enzymes. Recently, a novel metabolic function for HPS and PHI has been proposed in which these enzymes catalyze the reverse reaction for the biosynthesis of pentose phosphate in some archaeal strains. Thus the enzyme system plays a different role in bacteria and archaea by catalyzing the forward and reverse reactions respectively.

A new arrangement of (beta/alpha)8 barrels in the synthase subunit of PLP synthase.

Pyridoxal 5'-phosphate (PLP, vitamin B6), a cofactor in many enzymatic reactions, has two distinct biosynthetic routes, which do not coexist in any organism. Two proteins, known as PdxS and PdxT, together form a PLP synthase in plants, fungi, archaea, and some eubacteria. PLP synthase is a heteromeric glutamine amidotransferase in which PdxT produces ammonia from glutamine and PdxS combines ammonia with five- and three-carbon phosphosugars to form PLP. In the 2.2-A crystal structure, PdxS is a cylindrical dodecamer of subunits having the classic (beta/alpha)8 barrel fold. PdxS subunits form two hexameric rings with the active sites positioned on the inside. The hexamer and dodecamer forms coexist in solution. A novel phosphate-binding site is suggested by bound sulfate. The sulfate and another bound molecule, methyl pentanediol, were used to model the substrate ribulose 5-phosphate, and to propose catalytic roles for residues in the active site. The distribution of conserved surfaces in the PdxS dodecamer was used to predict a docking site for the glutaminase partner, PdxT.

Evolution of enzymatic activities in the orotidine 5'-monophosphate decarboxylase suprafamily: enhancing the promiscuous D-arabino-hex-3-ulose 6-phosphate synthase reaction catalyzed by 3-keto-L-gulonate 6-phosphate decarboxylase.

3-Keto-l-gulonate 6-phosphate decarboxylase (KGPDC) and d-arabino-hex-3-ulose 6-phosphate synthase (HPS) are members of the orotidine 5'-monophosphate decarboxylase (OMPDC) suprafamily [Wise, E., Yew, W. S., Babbitt, P. C., Gerlt, J. A., and Rayment, I. (2002) Biochemistry 41, 3861-3869], a group of homologous enzymes that share the (beta/alpha)(8)-barrel fold. KGPDC catalyzes a Mg(2+)-dependent decarboxylation reaction in the catabolic pathway of l-ascorbate utilization by Escherichia coli K-12 [Yew, W. S., and Gerlt, J. A. (2002) J.Bacteriol. 184, 302-306]; HPS catalyzes a Mg(2+)-dependent aldol condensation between formaldehyde and d-ribulose 5-phosphate in formaldehyde-fixing methylotrophic bacteria [Kato, N., Ohashi, H., Hori, T., Tani, Y., and Ogata, K. (1977) Agric. Biol. Chem. 41, 1133-1140]. Our previous studies of the KGPDC from E. coli established the occurrence of a stabilized cis-enediolate intermediate [Yew, W. S., Wise, E., Rayment, I., and Gerlt, J. A. (2004) Biochemistry 43, 6427-6437; Wise, E., Yew, W. S., Gerlt, J. A., and Rayment, I. (2004) Biochemistry 43, 6438-6446]. Although the mechanism of the HPS-catalyzed reaction has not yet been investigated, it also is expected to involve a Mg(2+)-stabilized cis-enediolate intermediate. We now have discovered that the KGPDC from E. coli and the HPS from Methylomonas aminofaciens are both naturally promiscuous for the reaction catalyzed by the homologue. On the basis of the alignment of the sequences of orthologous KGPDC's and HPS's, four conserved active site residues in the KGPDC from E. coli were mutated to those conserved in HPS's (E112D/R139V/T169A/R192A): the value of the k(cat) for the promiscuous HPS activity was increased as much as 170-fold (for the E112D/R139V/T169A/R192A mutant), and the value of k(cat)/K(m) was increased as much as 260-fold (for the E112D/R139V/T169A mutant); in both cases, the values of the kinetic constants for the natural KGPDC activity were decreased. Together with the structures of mutants reported in the accompanying manuscript [Wise, E. L., Yew, W. S., Akana, J., Gerlt, J. A., and Rayment, I., accompanying manuscript], these studies illustrate that large changes in catalytic efficiency can be accomplished with only modest changes in active site structure. Thus, the (beta/alpha)(8)-barrel fold shared by members of the OMPDC suprafamily appears well-suited for the evolution of new functions.

Evolution of enzymatic activities in the orotidine 5'-monophosphate decarboxylase suprafamily: structural basis for catalytic promiscuity in wild-type and designed mutants of 3-keto-L-gulonate 6-phosphate decarboxylase.

3-Keto-L-gulonate 6-phosphate decarboxylase (KGPDC) and D-arabino-hex-3-ulose 6-phosphate synthase (HPS), members of the orotidine 5'-monophosphate decarboxylase (OMPDC) suprafamily, catalyze reactions that involve the formation of Mg(2+)-ion stabilized 1,2-enediolate intermediates. The active sites of KGPDC and HPS share several conserved residues, including the presumed ligands for the Mg(2+) and a catalytic histidine residue that has been implicated in protonation of the intermediate in the KGPDC-catalyzed reaction. As reported in the previous manuscript, both enzymes are naturally promiscuous, with KGPDC from Escherichia coli catalyzing a low level of the HPS reaction and the HPS from Methylomonas aminofaciens catalyzing a significant level of the KGPDC reaction. Interestingly, the promiscuous HPS reaction catalyzed by KGPDC can be significantly enhanced by replacing no more than four active site residues from KGPDC reaction with residues from HPS. In this manuscript, we report structural studies of wild-type and mutant KDGPC's that provide a structural explanation for both the natural promiscuity for the HPS reaction and the enhanced HPS activity and diminished KGPDC activity catalyzed by active site mutants.