Dissociative phosphoryl transfer in PEP mutase catalysis: structure of the enzyme/sulfopyruvate complex and kinetic properties of mutants

The crystal structure of PEP mutase from Mytilus edulis in complex with a substrate-analogue inhibitor, sulfopyruvate S-pyr (K(i) = 22 microM), has been determined at 2.25 A resolution. Mg(II)-S-pyr binds in the alpha/beta barrel's central channel, at the C-termini of the beta-strands. The binding mode of S-pyr's pyruvyl moiety resembles the binding mode of oxalate seen earlier. The location of the sulfo group of S-pyr is postulated to mimic the phosphonyl group of the product phosphonopyruvate (P-pyr). This sulfo group interacts with the guanidinium group of Arg159, but it is not aligned for nucleopilic attack by neighboring basic amino side chains. Kinetic analysis of site directed mutants, probing the key active site residues Asp58, Arg159, Asn122, and His190 correlate well with the structural information. The results presented here rule out a phosphoryl transfer mechanism involving a double displacement, and suggest instead that PEP mutase catalysis proceeds via a dissociative mechanism in which the pyruvyl C(3) adds to the same face of the phosphorus from which the C(2)O departs. We propose that Arg159 and His190 serve to hold the phosphoryl/metaphosphate/phosphonyl group stationary along the reaction pathway, while the pyruvyl C(1)-C(2) bond rotates upon formation of the metaphosphate. In agreement with published data, the phosphoryl group transfer occurs on the Si-face of PEP with retention of configuration at phosphorus.     

Unraveling a bacterial hexose transport pathway

Structural information about proteins involved in bacterial hexose transport mediated by the phosphoenolpyruvate:sugar phosphotransferase system is rapidly accumulating. Within the past year, two crystal structures and two solution NMR structures of the histidine-containing phosphocarrier protein have been reported, adding structural details to previous NMR and crystallographic work on this protein and on enzyme IIA. The crystal structure of the regulatory complex between the glucose enzyme IIA and glycerol kinase has been determined, and the association of the histidine-containing phosphocarrier protein and either the glucose enzyme IIA or the mannitol enzyme IIA have been studied by NMR. Proposals concerning the mechanism of phosphoryl transfer and the protein-protein interactions involved may now be tested more rigorously using these data.     

Structural analysis of denaturant-protein interactions: Comparison between the effects of bromoethanol and SDS on denaturation and renaturation of triclinic lysozyme

This paper summarizes our crystallographic studies of the interaction of denaturants with cross-linked triclinic lysozyme. Electron density maps of various bromoethanol-lysozyme complexes are analyzed and compared to those reported earlier for SDS-lysozyme complexes. Despite differences in the chemical nature and size of the two denaturants their mode of interaction with the protein is quite similar, suggesting the existence of a general mechanism for binding of hydrophobic-hydrophilic denaturants to proteins. Our results are consistent with the conclusion that lysozyme consists of two domains connected by a flexible segment and that this segment represents an internal degree of freedom of the protein.The work was carried out during the tenure of a fellowship from the European Molecular Biology OrganizationWe are grateful to Dr. Gerson Cohen for providing us with his data processing programs, to Drs. David Haas, Paul Sigler, Thomas Creighton and Micael James for helpful discussions, and to Mr. Samuel Getteno for his invaluable technical assistance.     

The parental investment conflict in continuous time: St. Peter's fish as an example

The parental investment conflict considers the question of how much each sex should invest in each brood, thereby characterizing different animal species. Each species usually adopts a certain parental care pattern: female-care only, male-care only, biparental care, or even no parental care at all. The differences in care patterns are usually explained by the different costs and benefits arising from caring for the offspring in each animal species. This paper proposes a game-theoretical model to the parental investment conflict based on the parental behavior of St. Peter's fish. St. Peter's fish exhibit different parental care patterns, allowing the examination of the factors which determine the particular behavior in each mating. We present a continuous time, two-stage, asymmetric game, with two types of players: male and female. According to the model's results, three parental care patterns: male-only care, female-only care and biparental care, are possible evolutionarily stable strategies. The evolutionarily stable parental care pattern in a certain mating depends on a parent's increase in mortality due to parental care, and on its advantage from biparental care. These results may explain the different parental care patterns observed in a variety of animal species, including those found in the St. Peter's fish.     

Crystal structure of the petal death protein from carnation flower

Expression of the PSR132 protein from Dianthus caryophyllus (carnation, clover pink) is induced in response to ethylene production associated with petal senescence, and thus the protein is named petal death protein (PDP). Recent work has established that despite the annotation of PDP in sequence databases as carboxyphosphoenolpyruvate mutase, the enzyme is actually a C-C bond cleaving lyase exhibiting a broad substrate profile. The crystal structure of PDP has been determined at 2.7 A resolution, revealing a dimer-of-dimers oligomeric association. Consistent with sequence homology, the overall alpha/beta barrel fold of PDP is the same as that of other isocitrate lyase/PEP mutase superfamily members, including a swapped eighth helix within a dimer. Moreover, Mg(2+) binds in the active site of PDP with a coordination pattern similar to that seen in other superfamily members. A compound, covalently bound to the catalytic residue, Cys144, was interpreted as a thiohemiacetal adduct resulting from the reaction of glutaraldehyde used to cross-link the crystals. The Cys144-carrying flexible loop that gates access to the active site is in the closed conformation. Models of bound substrates and comparison with the closed conformation of isocitrate lyase and 2-methylisocitrate lyase revealed the structural basis for the broad substrate profile of PDP.     

Crystal structures of 2-methylisocitrate lyase in complex with product and with isocitrate inhibitor provide insight into lyase substrate specificity, catalysis and evolution

Two crystal structures of the C123S mutant of 2-methylisocitrate lyase have been determined, one with the bound reaction products, Mg(2+)-pyruvate and succinate, and the second with a bound Mg(2+)-(2R,3S)-isocitrate inhibitor. Comparison with the structure of the wild-type enzyme in the unbound state reveals that the enzyme undergoes a conformational transition that sequesters the ligand from solvent, as previously observed for two other enzyme superfamily members, isocitrate lyase and phosphoenolpyruvate mutase. The binding modes reveal the determinants of substrate specificity and stereoselectivity, and the stringent specificity is verified in solution using various potential substrates. A model of bound 2-methylisocitrate has been developed based on the experimentally determined structures. We propose a catalytic mechanism involving an alpha-carboxy-carbanion intermediate/transition state, which is consistent with previous stereochemical experiments showing inversion of configuration at the C(3) of 2-methylisocitrate. Structure-based sequence analysis and phylogenic tree construction reveal determinants of substrate specificity, highlight nodes of divergence of families, and predict enzyme families with new functions.     

From structure to function: YrbI from Haemophilus influenzae (HI1679) is a phosphatase

The crystal structure of the YrbI protein from Haemophilus influenzae (HI1679) was determined at a 1.67-A resolution. The function of the protein had not been assigned previously, and it is annotated as hypothetical in sequence databases. The protein exhibits the alpha/beta-hydrolase fold (also termed the Rossmann fold) and resembles most closely the fold of the L-2-haloacid dehalogenase (HAD) superfamily. Following this observation, a detailed sequence analysis revealed remote homology to two members of the HAD superfamily, the P-domain of Ca(2+) ATPase and phosphoserine phosphatase. The 19-kDa chains of HI1679 form a tetramer both in solution and in the crystalline form. The four monomers are arranged in a ring such that four beta-hairpin loops, each inserted after the first beta-strand of the core alpha/beta-fold, form an eight-stranded barrel at the center of the assembly. Four active sites are located at the subunit interfaces. Each active site is occupied by a cobalt ion, a metal used for crystallization. The cobalt is octahedrally coordinated to two aspartate side-chains, a backbone oxygen, and three solvent molecules, indicating that the physiological metal may be magnesium. HI1679 hydrolyzes a number of phosphates, including 6-phosphogluconate and phosphotyrosine, suggesting that it functions as a phosphatase in vivo. The physiological substrate is yet to be identified; however the location of the gene on the yrb operon suggests involvement in sugar metabolism.     

Distribution of Glutamate Transporter Subtypes During Human Brain Development

Abstract: In the mature brain, removal of glutamate from the synaptic cleft plays an important role in the maintenance of subtoxic levels of glutamate. This requirement is handled by a family of glutamate transporters, EAAT1, EAAT2, EAAT3, and EAAT4. Due to the involvement of glutamate also in neuronal development, it is believed that glutamate transport plays a role in developmental processes as well. Therefore, we have used immunohistochemical and immunoblot analysis to determine the distribution of the four glutamate transporters during human brain development using human pre- and postnatal brain tissue. Regional analysis showed that each transporter subtype has a unique distribution during development. EAAT2 was the most prominent glutamate transporter subtype and was highly enriched in cortex, basal ganglia, cerebellum, and thalamus in all ages examined. EAAT1 immunoreactivity was lower than that of EAAT2, with predominant localization in cortex, basal ganglia, hippocampus, and periventricular region. EAAT3 was located mainly in cortex, basal ganglia, and hippocampus, and EAAT4 was found only in cortex, hippocampus, and cerebellar cortex. The distinct regional distribution of various EAAT subtypes and also the transient expression of specific EAAT subtypes during development suggest multiple functional roles for glutamate transporters in the developing brain.