The structure of L-ribulose-5-phosphate 4-epimerase: an aldolase-like platform for epimerization.

The structure of L-ribulose-5-phosphate 4-epimerase from E. coli has been solved to 2.4 A resolution using X-ray diffraction data. The structure is homo-tetrameric and displays C(4) symmetry. Each subunit has a single domain comprised of a central beta-sheet flanked on either side by layers of alpha-helices. The active site is identified by the position of the catalytic zinc residue and is located at the interface between two adjacent subunits. A remarkable feature of the structure is that it shows a very close resemblance to that of L-fuculose-1-phosphate aldolase. This is consistent with the notion that both enzymes belong to a superfamily of epimerases/aldolases that catalyze carbon-carbon bond cleavage reactions via a metal-stabilized enolate intermediate. Detailed inspection of the epimerase structure, however, indicates that despite the close overall structural similarity to class II aldolases, the enzyme has evolved distinct active site features that promote its particular chemistry.

Catalysis and binding in L-ribulose-5-phosphate 4-epimerase: a comparison with L-fuculose-1-phosphate aldolase.

L-Ribulose-5-phosphate (L-Ru5P) 4-epimerase and L-fuculose-1-phosphate (L-Fuc1P) aldolase are evolutionarily related enzymes that display 26% sequence identity and a very high degree of structural similarity. They both employ a divalent cation in the formation and stabilization of an enolate during catalysis, and both are able to deprotonate the C-4 hydroxyl group of a phosphoketose substrate. Despite these many similarities, subtle distinctions must be present which allow the enzymes to catalyze two seemingly different reactions and to accommodate substrates differing greatly in the position of the phosphate (C-5 vs C-1). Asp76 of the epimerase corresponds to the key catalytic acid/base residue Glu73 of the aldolase. The D76N mutant of the epimerase retained considerable activity, indicating it is not a key catalytic residue in this enzyme. In addition, the D76E mutant did not show enhanced levels of background aldolase activity. Mutations of residues in the putative phosphate-binding pocket of the epimerase (N28A and K42M) showed dramatically higher values of K(M) for L-Ru5P. This indicates that both enzymes utilize the same phosphate recognition pocket, and since the phosphates are positioned at opposite ends of the respective substrates, the two enzymes must bind their substrates in a reversed or "flipped" orientation. The epimerase mutant D120N displays a 3000-fold decrease in the value of k(cat), suggesting that Asp120' provides a key catalytic acid/base residue in this enzyme. Analysis of the D120N mutant by X-ray crystallography shows that its structure is indistinguishable from that of the wild-type enzyme and that the decrease in activity was not simply due to a structural perturbation of the active site. Previous work [Lee, L. V., Poyner, R. R., Vu, M. V., and Cleland, W. W. (2000) Biochemistry 39, 4821-4830] has indicated that Tyr229' likely provides the other catalytic acid/base residue. Both of these residues are supplied by an adjacent subunit. Modeling of L-Ru5P into the active site of the epimerase structure suggests that Tyr229' is responsible for deprotonating L-Ru5P and Asp120' is responsible for deprotonating its epimer, D-Xu5P.

Active site residues of glutamate racemase.

Glutamate racemase, MurI, catalyzes the interconversion of glutamate enantiomers in a cofactor-independent fashion and provides bacteria with a source of D-Glu for use in peptidoglycan biosynthesis. The enzyme uses a "two-base" mechanism involving a deprotonation of the substrate at the alpha-position to form an anionic intermediate, followed by a reprotonation in the opposite stereochemical sense. In the Lactobacillus fermenti enzyme, Cys73 is responsible for the deprotonation of D-glutamate, and Cys184 is responsible for the deprotonation of L-glutamate; however, very little is known about the roles of other active site residues. This work describes the preparation of four mutants in which strictly conserved residues containing ionizable side chains were modified (D10N, D36N, E152Q, and H186N). During the course of this research, the structural analysis of a crystallized glutamate racemase indicated that three of these residues (D10, E152, and H186) are in the active site of the enzyme [Hwang, K. Y., Cho, C.-S., Kim, S. S., Sung, H.-C., Yu, Y. G., and Cho, Y. (1999) Nat. Struct. Biol. 6, 422-426]. Two of the mutants, D10N and H186N, displayed a marked decrease in the values of k(cat), but not K(M), and are therefore implicated as important catalytic residues. Further analysis of the primary kinetic isotope effects observed with alpha-deuterated substrates showed that a significant asymmetry was introduced into the free energy profile by these two mutations. This is interpreted as evidence that the mutated residues normally assist the catalytic thiols in acting as bases (D10 with C73 and H186 with C184). An alternate possibility is that the residues may serve to stabilize the carbanionic intermediate in the racemization reaction.

The structure of UDP-N-acetylglucosamine 2-epimerase reveals homology to phosphoglycosyl transferases.

Bacterial UDP-N-acetylglucosamine 2-epimerase catalyzes the reversible epimerization at C-2 of UDP-N-acetylglucosamine (UDP-GlcNAc) and thereby provides bacteria with UDP-N-acetylmannosamine (UDP-ManNAc), the activated donor of ManNAc residues. ManNAc is critical for several processes in bacteria, including formation of the antiphagocytic capsular polysaccharide of pathogens such as Streptococcus pneumoniae types 19F and 19A. We have determined the X-ray structure (2.5 A) of UDP-GlcNAc 2-epimerase with bound UDP and identified a previously unsuspected structural homology with the enzymes glycogen phosphorylase and T4 phage beta-glucosyltransferase. The relationship to these phosphoglycosyl transferases is very intriguing in terms of possible similarities in the catalytic mechanisms. Specifically, this observation is consistent with the proposal that the UDP-GlcNAc 2-epimerase-catalyzed elimination and re-addition of UDP to the glycal intermediate may proceed through a transition state with significant oxocarbenium ion-like character. The homodimeric epimerase is composed of two similar alpha/beta/alpha sandwich domains with the active site located in the deep cleft at the domain interface. Comparison of the multiple copies in the asymmetric unit has revealed that the epimerase can undergo a 10 degrees interdomain rotation that is implicated in the regulatory mechanism. A structure-based sequence alignment has identified several basic residues in the active site that may be involved in the proton transfer at C-2 or stabilization of the proposed oxocarbenium ion-like transition state. This insight into the structure of the bacterial epimerase is applicable to the homologous N-terminal domain of the bifunctional mammalian UDP-GlcNAc "hydrolyzing" 2-epimerase/ManNAc kinase that catalyzes the rate-determining step in the sialic acid biosynthetic pathway.

The first structure of UDP-glucose dehydrogenase reveals the catalytic residues necessary for the two-fold oxidation.

Bacterial UDP-glucose dehydrogenase (UDPGlcDH) is essential for formation of the antiphagocytic capsule that protects many virulent bacteria such as Streptococcus pyogenes andStreptococcus pneumoniae type 3 from the host's immune system. We have determined the X-ray structures of both native and Cys260Ser UDPGlcDH from S. pyogenes (74% similarity to S. pneumoniae) in ternary complexes with UDP-xylose/NAD(+) and UDP-glucuronic acid/NAD(H), respectively. The 402 residue homodimeric UDPGlcDH is composed of an N-terminal NAD(+) dinucleotide binding domain and a C-terminal UDP-sugar binding domain connected by a long (48 A) central alpha-helix. The first 290 residues of UDPGlcDH share structural homology with 6-phosphogluconate dehydrogenase, including conservation of an active site lysine and asparagine that are implicated in the enzyme mechanism. Also proposed to participate in the catalytic mechanism are a threonine and a glutamate that hydrogen bond to a conserved active site water molecule suitably positioned for general acid/base catalysis.

Catalytic acid/base residues of glutamate racemase.

Glutamate racemase is a cofactor-independent enzyme that employs two active-site cysteine residues as acid/base catalysts during the interconversion of glutamate enantiomers. In a given reaction direction, a thiolate from one of the cysteines abstracts the alpha-proton, and the other cysteine thiol delivers a proton to the opposite face of the resulting carbanionic intermediate. This paper reports that the C73S and C184S mutants are still capable of racemizing glutamate with specificity constants about 10(3)-fold lower than those of the wild-type enzyme. A "one-base requiring" reaction, the elimination of water from N-hydroxyglutamate, has been used to deduce which thiol acts as the base for a given enantiomer. With D-N-hydroxyglutamate the C73S mutant is a much poorer catalyst than wild-type enzyme, whereas the C184S mutant is a somewhat better catalyst. This trend was reversed with L-N-hydroxyglutamate, suggesting that Cys73 is responsible for the deprotonation of D-glutamate and Cys184 is responsible for the deprotonation of L-glutamate. In addition, with C73S the Vmax/KM isotope effect on D-glutamate racemization was greater than that seen with wild-type enzyme, whereas the isotope effect with L-glutamate had decreased. The results were reversed with the C184S mutant. This is interpreted as being due to an asymmetry in the free energy profiles that is induced upon mutation, with the deprotonation step involving a serine becoming the more cleanly rate-determining of the two. These results support the above assignment and the notion that a carbanionic intermediate is formed during catalysis.

UDP-N-trifluoroacetylglucosamine as an alternative substrate in N-acetylglucosaminyltransferase reactions.

The synthesis of UDP-N-trifluoroacetylglucosamine [uridine 5'-(2-trifluoroacetamido-2-deoxy-alpha-D-glucopyranosyl diphosphate, UDP-GlcNAc-F3] is reported. The compound is found to serve as a substrate for the core-2 GlcNAc transferase (EC 2.4.1.102) that is involved in the biosynthesis of O-linked glycoproteins and for the GlcNAcT-V transferase (EC 2.4.1.155) that is a key biosynthetic enzyme controlling the branching pattern of cell surface complex Asn-linked oligosaccharides. The trisaccharide beta-D-Galp-(1-->3) -[beta-D-GlcpNAc-F3(1-->6)] alpha-D-GalpNAc-OR [R = (CH2)8CO2Me] was prepared from beta-D-Galp-(1-->3) -alpha-D- GalpNAc-OR using the core-2 GlcNAc transferase. The tetrasaccharide beta-D-GlcpNAc-(1 -->2)-[beta-D-GlcpNAc-F3-(1-->6)]-alpha-D-Manp-(1-->6)-beta-D-Glcp -OR [R = (CH2)7CH3] using the GlcNAcT-V transferase. Removal of the trifluoroacetyl group was achieved under mild basic conditions to give the corresponding glucosamine containing tetrasaccharide. These examples demonstrate the feasibility of introducing masked forms of glucosamine residues into oligosaccharides using GlcNAc-specific transferases. The requirement for the trifluoroacetamido group as a specific recognition element was evident in the observation that neither UDP-glucosamine nor UDP-glucose served as a donor substrates for the core-2 GlcNAc transferase.

Epimerization via carbon-carbon bond cleavage. L-ribulose-5-phosphate 4-epimerase as a masked class II aldolase.

Studies indicating that the E. coli L-ribulose-5-phosphate 4-epimerase employs an "aldolase-like" mechanism are reported. This NAD+-independent enzyme epimerizes a stereocenter that does not bear an acidic proton and therefore it cannot utilize a simple deprotonation-reprotonation mechanism. Sequence similarities between the epimerase and the class II l-fuculose-1-phosphate aldolase suggest that the two may be evolutionarily related and that the epimerization may occur via carbon-carbon bond cleavage and re-formation. Conserved residues thought to provide the metal ion ligands of the epimerase have been modified using site-directed mutagenesis. The resulting mutants show low kcat values in addition to a reduced affinity for Zn2+. These observations serve to establish that there is a structural link between between the active site geometry of the epimerase and the aldolase. In addition, the H97N mutant was found to catalyze the condensation of dihydroxyacetone and glycolaldehyde phosphate to produce a mixture of L-ribulose-5-phosphate and D-xylulose-5-phosphate. This observation of aldolase activity establishes that the epimerase active site is capable of promoting carbon-carbon bond cleavage. Furthermore, glycolaldehyde phosphate was shown to be a competitive inhibitor of the mutant enzyme (KI = 0.37 mM) but not of the wild-type enzyme. The mutation apparently causes the epimerase to become "leaky" and enables it to bind/generate the normal reaction intermediates from the unbound aldol cleavage products.

Health, growth, and use of community services in NICU graduates at early school age.

PURPOSE: To examine outcomes related to health, growth, and use of community health and education services in children ages 6 to 8 years who received newborn intensive care because of prematurity or perinatal complications. METHOD: Parents of 81 children who had received neonatal intensive care at a Midwest US tertiary care center completed a mailed questionnaire. Three birth weight groups (very low birth weight [VLBW] < 1500 g, n = 35; low birth weight [LBW] 1501-2500 g, n = 24, and normal birth weight [NBW] > 2500 g, n = 22) were compared regarding growth, health, and use of community-based services using descriptive statistics and one-way analysis of variance. FINDINGS: VLBW and NBW groups had more ongoing health concerns. Growth patterns were similar in all groups. VLBW and NBW groups demonstrated greater use of community-based services, and service use increased at school age. CONCLUSIONS: Comprehensive systems are needed for follow-up of high-risk infants to detect and refer problems early. Neonatal histories must be tracked throughout childhood. Seriously ill term NBW infants are at risk for later morbidity and require follow-up similar to that provided for VLBW children.

Properties and kinetic analysis of UDP-glucose dehydrogenase from group A streptococci. Irreversible inhibition by UDP-chloroacetol.

UDP-glucuronic acid is used by many pathogenic bacteria in the construction of an antiphagocytic capsule that is required for virulence. The enzyme UDP-glucose dehydrogenase catalyzes the NAD+-dependent 2-fold oxidation of UDP-glucose and provides a source of the acid. In the present study the recombinant dehydrogenase from group A streptococci has been purified and found to be active as a monomer. The enzyme contains no chromophoric cofactors, and its activity is unaffected by the presence of EDTA or carbonyl-trapping reagents. Initial velocity and product inhibition kinetic patterns are consistent with a bi-uni-uni-bi ping-pong mechanism in which UDP-glucose is bound first and UDP-glucuronate is released last. UDP-xylose was found to be a competitive inhibitor (Ki, 2.7 microM) of the enzyme. The enzyme is irreversibly inactivated by uridine 5'-diphosphate-chloroacetol due to the alkylation of an active site cysteine thiol. The apparent second order rate constant for the inhibition (ki/Ki) was found to be 2 x 10(3) mM-1 min-1. Incubation with the truncated compound, chloroacetol phosphate, resulted in no detectable inactivation when tested under comparable conditions. This supports the notion that uridine 5'-diphosphate-chloroacetol is bound in the place of UDP-glucose and is not simply acting as a nonspecific alkylating agent.

Study of the reaction mechanism of the D-glutamic acid-adding enzyme from Escherichia coli.

The D-glutamic acid-adding enzyme of Escherichia coli, or MurD, was purified from an overproducing strain and a few aspects of its reaction mechanism were studied. The existence of a reactive cysteinyl residue was shown by the following experiments: (1) two thiol-modifying reagents, (5,5'-dithiobis)2-nitrobenzoic acid and 2-nitro-5-thiocyanobenzoic acid, inactivated the enzyme; (2) incubation with tetranitromethane led to inactivation and to the appearance of cysteic acid (not to 3-nitrotyrosine); (3) in each case, ATP or UDP-MurNAc-L-Ala (but not D-glutamic acid) protected the enzyme from inactivation. The existence of a reactive lysyl residue was shown by the action of 2,4,6-trinitrobenzenesulfonic acid, a reagent specific for lysyl residues present in phosphate-binding sites. The formation of an acyl phosphate intermediate was consistent with three types of results: (1) the molecular isotope exchange reaction, which took place only in the presence of phosphate, but which was not strictly dependent on the presence of ADP; (2) a release of phosphate, measured by the molybdate assay, observed when the enzyme was incubated with ATP and UDP-MurNAc-L-Ala (without D-glutamic acid); (3) the appearance of a new radioactive compound (besides ATP and Pi) after incubation for a few minutes with UDP-MurNAc-L-Ala and [gamma-32P]ATP. Finally, the fact that phosphinate 1 was a good inhibitor of the enzyme (IC50 = 0.7 microM) strongly suggested that a tetrahedral transition state follows the acyl phosphate in the reaction pathway.

Mechanism of the reaction catalyzed by glutamate racemase.

The mechanism of the cofactor-independent glutamate racemase from Lactobacillus has been studied. The possible formation of an acylenzyme intermediate during catalysis has been investigated using 18O-carboxyl labeled glutamate. The absence of any washout of label during racemization argues against intermediate formation. The observation of the enzyme-catalyzed incorporation of deuterium at the C-2 position of glutamate upon racemization in D2O provides evidence for a deprotonation/protonation mechanism. Further experiments have been performed in order to determine the number of enzymic bases responsible for racemization. Solvent deuterium is efficiently incorporated into the product enantiomer but not into the recovered substrate enantiomer in each reaction direction. This finding is consistent with a "two-base" mechanism in which one enzymic base deprotonates the substrate, and the conjugate acid of a second enzymic base protonates the resulting intermediate from the opposite face. It also suggests that the two bases are monoprotic. The possibility that the two enzymic forms, which differ at the very least by the protonation states of the active-site bases, are kinetically significant has been examined by measuring the entire time course of the approach to equilibrium at various concentrations of glutamate. An "oversaturated" regime [Fisher, L. M., Albery, W. J., & Knowles, J. R. (1986) Biochemistry 25, 2529-2537] was not observed using glutamate concentrations as high as 100 mM, indicating that the two enzyme forms are rapidly interconverting under physiological conditions.

Isotope effects and the identification of catalytic residues in the reaction catalyzed by glutamate racemase.

Primary kinetic isotope effects on Vmax were observed in both reaction directions upon racemizing samples of [2-2H]glutamate with the cofactor-independent glutamate racemase from Lactobacillus. This supports a deprotonation/protonation mechanism for racemization in which the breaking of the carbon-hydrogen bond at C-2 is partially rate-determining. Substantial "overshoots" were observed when the time course of racemization of either enantiomer of glutamate was monitored using circular dichroism spectroscopy. This is consistent with a "two-base" mechanism accompanied by a kinetic isotope effect. "Competitive deuterium washout" experiments were used to measure kinetic isotope effects on Vmax/Km of 2.5 for (S)-glutamate and 3.4 for (R)-glutamate. The ratio of the notably different isotope effects was confirmed by "double competitive deuterium washout" experiments. Site-directed mutagenesis was used to generate the mutant C73A and C184A enzymes. In each case the mutant enzymes were inactive as racemases. The two mutant enzymes are, however, capable of catalyzing the elimination of HCl from opposite enantiomers of threo-3-chloroglutamic acid, a process that presumably requires only one enzymic base. This finding indicates that the active sites of the mutant enzymes are intact and that the two cysteines flank the bound substrate molecule. It appears that cysteine-73 is responsible for the abstraction of the C-2 hydrogen from (R)-glutamate and cysteine-184 abstracts the proton from (S)-glutamate in the racemization reaction of the wild-type enzyme.

Polyvalent human immunodeficiency virus synthetic immunogen comprised of envelope gp120 T helper cell sites and B cell neutralization epitopes.

In previous studies, we have used antisera raised to envelope (env)-gene-encoded synthetic peptides to identify a region of (HIV) glycoprotein (gp) 120 env protein designated SP10 that contains a type-specific neutralizing determinant. To develop a polyvalent, synthetic peptide inoculum that can evoke both neutralizing antibodies and T cell proliferative responses to more than one HIV isolate, synthetic peptides containing type-specific neutralizing determinants of gp120 from HIV isolates HTLV-IIIB (IIIB), HTLV-IIIMN (MN) and HTLV-IIIRF (RF) were coupled to a 16 amino acid T cell epitope (T1) of HIV-IIIB gp120 and used to immunize goats. Goat antisera to each T1-SP10 peptide derived from the SP10 region of gp120 of IIIB, MN, and RF neutralized HIV isolates IIIB, MN and RF in a type-specific manner. Moreover, peripheral blood T cells from immunized goats also proliferated in a type-specific manner to peptides derived from gp120 of IIIB, MN, and RF. When combined in a trivalent inoculum, T1-SP10 peptides from HIV-1 isolates IIIB, MN, and RF evoked a high titered neutralizing antibody response to isolates IIIB, MN, and RF in goats and as well induced immune T cells to undergo blast transformation in the presence of peptides derived from gp120 of all three HIV isolates. The T1 portion of the T1-SP10 construct was shown to induce a B cell antibody response against determinants within the T1 peptide in addition to inducing T cell proliferative responses in immune goat T cells. Moreover, the SP10 portion of the T1-SP10 constructs not only induced B cell antibody production but also induced type-specific T cell proliferative responses localized to the C-terminal variable sequences of the SP10 peptides. Finally, the T1-SP10 peptide construct induced memory T cell proliferative responses to native gp120 env protein. Thus, combinations of homologous SP10 region synthetic peptides containing type-specific neutralizing determinants and T cell epitopes of HIV gp120 may be useful in man to elicit high titered neutralizing B cell responses and, as well, T cell responses to more than one HIV isolate.

Mapping of immunogenic regions of human T cell leukemia virus type I (HTLV-I) gp46 and gp21 envelope glycoproteins with env-encoded synthetic peptides and a monoclonal antibody to gp46.

Antigenic sites on human T cell leukemia virus type I (HTLV-I) gp46 and gp21 envelope glycoproteins that are immunogenic in man were studied with envelope gene (env)-encoded synthetic peptides and a mAb to HTLV-I gp46 envelope glycoprotein. Antibodies in 78% of sera from HTLV-I seropositive subjects reacted with synthetic peptide 4A (amino acids 190 to 209) from a central region of HTLV-I gp46. Human anti-HTLV-I antibodies also bound to synthetic peptides 6 (29% of sera) and 7 (18% of sera) from a C-terminal region of gp46 (amino acids 296 to 312) and an N-terminal region of gp21 (amino acids 374 to 392), respectively. mAb 1C11 raised to affinity-purified HTLV-I gp46 reacted with gp46 external envelope glycoprotein and gp63 envelope precursor in immunoblot assay and also bound to the surface of HTLV-I+ cells lines HUT-102 and MT-2. Antibody 1C11 did not react with HTLV-II or HIV-infected cells or with a broad panel of normal human tissues or cell lines. In competitive RIA, anti-gp46 antibody 1C11 was inhibited from binding to gp46 either by antibodies from HTLV-I seropositive subjects or by HTLV-I env-encoded synthetic peptide 4A, indicating that 1C11 bound to or near a site on gp46 within amino acids 190 to 209 also recognized by antibodies from HTLV-I-seropositive individuals. When tested in syncytium inhibition assay, mAb 1C11 did not neutralize the infectivity of HTLV-I. Thus, HTLV-I infection in man is associated with a major antibody response to a region of gp46 within amino acids 190 to 209 that is on the surface of virus-infected cells.