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M H Saier

Publications and source records attributed to M H Saier.

At least 55 records · Page 3Linked to original sources
PubMed1999

Enzyme I(Ntr) from Escherichia coli. A novel enzyme of the phosphoenolpyruvate-dependent phosphotransferase system exhibiting strict specificity for its phosphoryl acceptor, NPr.

The phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) phosphorylates sugars and regulates cellular metabolic processes using a phosphoryl transfer chain including the general energy coupling proteins, Enzyme I (EI) and HPr as well as the sugar-specific Enzyme II complexes. Analysis of the Escherichia coli genome has revealed the presence of 5 paralogues of EI and 5 paralogues of HPr, most of unknown function. The ptsP gene encodes an EI paralogue designated Enzyme I(nitrogen) (EI(Ntr)), and two genes located in the rpoN operon encode PTS protein paralogues, NPr and IIA(Ntr), both implicated in the regulation of sigma(54) activity. The ptsP gene was polymerase chain reaction amplified from the E. coli chromosome and cloned into an overexpression vector allowing the overproduction and purification of EI(Ntr). EI(Ntr) was shown to phosphorylate NPr in vitro using either a [(32)P]PEP-dependent protein phosphorylation assay or a quantitative sugar phosphorylation assay. EI(Ntr) phosphorylated NPr but not HPr, whereas Enzyme I exhibited a strong preference for HPr. These two pairs of proteins (EI(Ntr)/NPr and EI/HPr) thus exhibit little cross-reactivity. Phosphoryl transfer from PEP to NPr catalyzed by EI(Ntr) has a pH optimum of 8.0, is dependent on Mg(2+), is stimulated by high ionic strength, and exhibits two K(m) values for NPr (2 and 10 microM) possibly because of negative cooperativity. The results suggest that E. coli possesses at least two distinct PTS phosphoryl transfer chains, EI(Ntr) --> NPr --> IIA(Ntr) and EI --> HPr --> IIA(sugar). Sequence comparisons allow prediction of residues likely to be important for specificity. This is the first report demonstrating specificity at the level of the energy coupling proteins of the PTS.

Amino Acid Sequence↗
PubMed1999

Phylogenetic characterization of novel transport protein families revealed by genome analyses.

As a result of recent genome sequencing projects as well as detailed biochemical, molecular genetic and physiological experimentation on representative transport proteins, we have come to realize that all organisms possess an extensive but limited array of transport protein types that allow the uptake of nutrients and excretion of toxic substances. These proteins fall into phylogenetic families that presumably reflect their evolutionary histories. Some of these families are restricted to a single phylogenetic group of organisms and may have arisen recently in evolutionary time while others are found ubiquitously and may be ancient. In this study we conduct systematic phylogenetic analyses of 26 families of transport systems that either had not been characterized previously or were in need of updating. Among the families analyzed are some that are bacterial-specific, others that are eukaryotic-specific, and others that are ubiquitous. They can function by either a channel-type or a carrier-type mechanism, and in the latter case, they are frequently energized by coupling solute transport to the flux of an ion down its electrochemical gradient. We tabulate the currently sequenced members of the 26 families analyzed, describe the properties of these families, and present partial multiple alignments, signature sequences and phylogenetic trees for them all.

Amino Acid Sequence↗
PubMed1999

The amino acid/auxin:proton symport permease family.

Amino acids and their derivatives are transported into and out of cells by a variety of permease types which comprise several distinct protein families. We here present a systematic analysis of a group of homologous transport proteins which together comprise the eukaryotic-specific amino acid/auxin permease (AAAP) family (TC #2. 18). In characterizing this family, we have (1) identified all sequenced members of the family, (2) aligned their sequences, (3) identified regions of striking conservation, (4) derived a family-specific signature sequence, and (5) proposed a topological model that appears to be applicable to all members of the family. We have also constructed AAAP family phylogenetic trees and dendrograms using six different programs that allow us to trace the evolutionary history of the family, estimate the relatedness of proteins from dissimilar organismal phyla, and evaluate the reliability of the different programs available for phylogenetic studies. The TREE and neighbor-joining programs gave fully consistent results while CLUSTAL W gave similar but non-identical results. Other programs gave less consistent results. The phylogenetic analyses reveal (1) that many plant AAAP family proteins arose recently by multiple gene duplication events that occurred within a single organism, (2) that some plant members of the family with strikingly different specificities diverged early in evolutionary history, and (3) that AAAP family proteins from fungi and animals diverged from the plant proteins long ago, possibly when animals, plants and fungi diverged from each other. The Neurospora protein nevertheless exhibits overlapping specificity with those found in plants. Preliminary evidence is presented suggesting that proteins of the AAAP family are distantly related to proteins of the large ubiquitous amino acid/polyamine/choline family (TC #2.3) as well as to those of two small bacterial amino acid transporter families, the ArAAP family (TC #2.42) and the STP family (TC #2.43).

Amino Acid Sequence↗
PubMed1999

A functional-phylogenetic system for the classification of transport proteins.

Twenty completely sequenced genomes of bacteria, archaea, and eukaryotes have been surveyed for the presence of genes encoding homologues of known solute transport proteins. These analyses and others have demonstrated the presence of nearly 250 families of sequence-related transporters. All such proteins have been classified according to the system we call the transporter classification system of the transport commission (TC). This short summary article describes the main features of this system; the families are presented in tabular form. Detailed information concerning these families and their constituent transporters is available on our web sites. J. Cell. Biochem. Suppls. 32/33:84-94, 1999.

Animals↗
PubMed1999

Eukaryotic transmembrane solute transport systems.

A comprehensive classification system for transmembrane molecular transporters has been proposed. This system is based on (i) mode of transport and energy-coupling mechanism, (ii) protein phylogenetic family, (iii) phylogenetic cluster, and (iv) substrate specificity. The proposed "Transport Commission" (TC) system is superficially similar to that implemented decades ago by the Enzyme Commission for enzymes, but it differs from the latter system in that it uses phylogenetic and functional data for classification purposes. Very few families of transporters include members that do not function exclusively in transport. Analyses reported reveal that channels, primary carriers, secondary carriers (uni-, sym-, and antiporters), and group translocators comprise distinct categories of transporters, and that transport mode and energy coupling are relatively immutable characteristics. By contrast, substrate specificity and polarity of transport are often readily mutable. Thus, with very few exceptions, a unified family of transporters includes members that function by a single transport mode and energy-coupling mechanism although a variety of substrates may be transported with either inwardly or outwardly directed polarity. The TC system allows cross-referencing according to substrates transported and protein sequence database accession numbers. Thus, familial assignments of newly sequenced transport proteins are facilitated. In this article I examine families of transporters that are eukaryotic specific. These families include (i) channel proteins, mostly from animals; (ii) facilitators and secondary active transport carriers; (iii) a few ATP-dependent primary active transporters; and (iv) transporters of unknown mode of action or energy-coupling mechanism. None of the several ATP-independent primary active transport energy-coupling mechanisms found in prokaryotes is represented within the eukaryotic-specific families. The analyses reported provide insight into transporter families that may have arisen in eukaryotes after the separation of eukaryotes from archaea and bacteria. On the basis of the reported analyses, it is suggested that the horizontal transfer of genes encoding transport proteins between eukaryotes and members of the other two domains of life occurred very infrequently during evolutionary history.

Biological Transport↗
PubMed1999

Genome archeology leading to the characterization and classification of transport proteins.

In the study of transmembrane transport, molecular phylogeny provides a reliable guide to protein structure, catalytic and noncatalytic transport mechanisms, mode of energy coupling and substrate specificity. It also allows prediction of the evolutionary history of a transporter family, leading to estimations of its age, source, and route of appearance. Phylogenetic analyses, therefore, provide a rational basis for the characterization and classification of transporters. A universal classification system has been described, based on both function and phylogeny, which has been designed to be applicable to all currently recognized and yet-to-be discovered transport proteins found in living organisms on Earth.

Carrier Proteins↗
PubMed1999

Phylogenetic analyses of proton-translocating transhydrogenases.

The proton-translocating nicotinamide nucleotide transhydrogenases (TH) provide a simple model for understanding chemically coupled transmembrane proton translocation. To further our understanding of TH structure-function relationships, we have identified all sequenced homologous of these vectorial enzymes and have conducted sequence comparison studies. The NAD-binding domains of TH are homologous to bacterial alanine dehydrogenases (ADH) and eukaryotic saccharopine dehydrogenases (SDH) as well as N5(carboxyethyl)-L-ornithine synthase of Lactococcus lactis and dipicolinate synthase of Bacillus subtilis. A multiple alignment, a phylogenetic tree, and two signature sequences for this family, designated the TH-ADH-SDH or TAS superfamily, have been derived. Additionally, the TH family has been characterized. Phylogenetic analyses suggested that these proteins have evolved without inter-system shuffling. However, interdomain splicing-fusion events have occurred during the evolution of several of these systems. Analyses of the multiple alignment for the TH family revealed that domain conservation occurs in the order: NADP-binding domain (domain III) > NAD-binding domain (domain I) > proton-translocating transmembrane domain (domain II). A topologic model for the proton-translocating transmembrane domain consistent with published data is presented, and a possible involvement of specific transmembrane alpha-helical segments in channel formation is suggested.

Amino Acid Oxidoreductases↗
PubMed1999

Mutations that confer resistance to 2-deoxyglucose reduce the specific activity of hexokinase from Myxococcus xanthus.

The glucose analog 2-deoxyglucose (2dGlc) inhibits the growth and multicellular development of Myxococcus xanthus. Mutants of M. xanthus resistant to 2dGlc, designated hex mutants, arise at a low spontaneous frequency. Expression of the Escherichia coli glk (glucokinase) gene in M. xanthus hex mutants restores 2dGlc sensitivity, suggesting that these mutants arise upon the loss of a soluble hexokinase function that phosphorylates 2dGlc to form the toxic intermediate, 2-deoxyglucose-6-phosphate. Enzyme assays of M. xanthus extracts reveal a soluble hexokinase (ATP:D-hexose-6-phosphotransferase; EC 2.7.1.1) activity but no phosphotransferase system activities. The hex mutants have lower levels of hexokinase activities than the wild type, and the levels of hexokinase activity exhibited by the hex mutants are inversely correlated with the ability of 2dGlc to inhibit their growth and sporulation. Both 2dGlc and N-acetylglucosamine act as inhibitors of glucose turnover by the M. xanthus hexokinase in vitro, consistent with the finding that glucose and N-acetylglucosamine can antagonize the toxic effects of 2dGlc in vivo.

Acetylglucosamine↗
PubMed1999

The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human disease and development proteins.

A previous report identified and classified a small family of gram-negative bacterial drug and heavy metal efflux permeases, now commonly referred to as the RND family (TC no. 2.6). We here show that this family is actually a ubiquitous superfamily with representation in all major kingdoms. We report phylogenetic analyses that define seven families within the RND superfamily as follows: (1) the heavy metal efflux (HME) family (gram negative bacteria), (2) the hydrophobe/amphiphile efflux-1 (HAE1) family (gram negative bacteria), (3) the nodulation factor exporter (NFE) family (gram negative bacteria), (4) the SecDF protein-secretion accessory protein (SecDF) family (gram negative and gram positive bacteria as well as archaea), (5) the hydrophobe/amphiphile efflux-2 (HAE2) family (gram positive bacteria), (6) the eukaryotic sterol homeostasis (ESH) family, and (7) the hydrophobe/amphiphile efflux-3 (HAE3) family (archaea and spirochetes). Functionally uncharacterized proteins were identified that are members of the RND superfamily but fall outside of these seven families. Some of the eukaryotic homologues function as enzymes and receptors instead of (or in addition to) transporters. The sizes and topological patterns exhibited by members of all seven families are shown to be strikingly similar, and statistical analyses establish common descent. Multiple alignments of proteins within each family allow derivation of family-specific signature sequences. Structural, functional, mechanistic and evolutionary implication of the reported results are discussed.

Amino Acid Sequence↗
PubMed1999

The major facilitator superfamily.

In 1998 we updated earlier descriptions of the largest family of secondary transport carriers found in living organisms, the major facilitator superfamily (MFS). Seventeen families of transport proteins were shown to comprise this superfamily. We here report expansion of the MFS to include 29 established families as well as five probable families. Structural, functional, and mechanistic features of the constituent permeases are described, and each newly identified family is shown to exhibit specificity for a single class of substrates. Phylogenetic analyses define the evolutionary relationships of the members of each family to each other, and multiple alignments allow definition of family-specific signature sequences as well as all well-conserved sequence motifs. The work described serves to update previous publications and allows extrapolation of structural, functional and mechanistic information obtained with any one member of the superfamily to other members with limitations determined by the degrees of sequence divergence.

Amino Acid Sequence↗
PubMed1999

Modular assembly of voltage-gated channel proteins: a sequence analysis and phylogenetic study.

Voltage-sensitive cation-selective ion channels of the voltage-gated ion channel (VGC) superfamily were examined by a combination of sequence alignment and phylogenetic tree construction procedures. Segments of the alpha-subunits of K+-selective channels homologous to the structurally elucidated KcsA channel of Streptomyces lividans were multiply aligned, and this alignment provided the database for computer-assisted structural analyses and phylogenetic tree construction. Similar analyses were conducted with the four homologous repeats of the alpha-subunits from representative Ca2+- and Na+-selective channels, as well as with the ensemble of K+, Ca2+ and Na+ channels. In both the single subunit of the K+ channels and the individual repeats of the Ca2+ and Na+ channels, the analyses suggest the occurrence of at least two tandemly arranged modules corresponding to the predicted voltage-sensor domain and the pore domain. The phylogenetic analyses reveal strict clustering of segments according to cation-selectivity and repeat unit. We surmise that the pore module of the prokaryotic K+ channel was the primordial polypeptide upon which other modules were superimposed during evolution in order to generate phenotypic diversity. These observations may prove applicable to all members of the VGC family yet to be discovered throughout the prokaryotic and eukaryotic kingdoms.

Amino Acid Sequence↗
PubMed1999

Novel phosphotransferase systems revealed by bacterial genome analysis: the complete repertoire of pts genes in Pseudomonas aeruginosa.

We herein describe all genes encoding constituents of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in the 6Mbp genome of the opportunistic human pathogen, Pseudomonas aeruginosa. Only four gene clusters were found to encode identifiable PTS homologues. These genes clusters encode novel multidomain proteins, two complete sugar-specific PTS phosphoryl transfer chains for the metabolism of fructose and N-acetylglucosamine, and a complex regulatory system that may function to coordinate carbon and nitrogen metabolism. No previously characterized organism has been shown to exhibit such a novel and restricted complement of PTS proteins.

Genome, Bacterial↗
PubMed1999

The LysE superfamily: topology of the lysine exporter LysE of Corynebacterium glutamicum, a paradyme for a novel superfamily of transmembrane solute translocators.

In Corynebacterium glutamicum the LysE carrier protein exhibits the unique function of exporting L-lysine. We here analyze the membrane topology of LysE, a protein of 236 amino acyl residues, using PhoA- and LacZ-fusions. The amino-terminal end of LysE is located in the cytoplasm whereas the carboxy-terminal end is found in the periplasm. Although 6 hydrophobic domains were identified based on hydropathy analyses, only five transmembrane spanning helices appear to be present. The additional hydrophobic segment may dip into the membrane or be surface localized. We show that LysE is a member of a family of proteins found, for example, in Escherichia coil, Bacillus subtilis, Mycobacterium tuberculosis and Helicobacter pylori. This family, which we have designated the LysE family, is distantly related to two additional protein families which we have designated the YahN and CadD families. These three families, the members of which exhibit similar sizes, hydropathy profiles, and sequence motifs comprise the LysE superfamily. Functionally characterized members of the LysE superfamily export L-lysine, cadmium and possibly quarternary amines. We suggest that LysE superfamily members will prove to catalyze export of a variety of biologically important solutes.

Amino Acid Sequence↗
PubMed1998

Sequence analyses and phylogenetic characterization of the ZIP family of metal ion transport proteins.

Several novel but similar heavy metal ion transporters, Zrt1, Zrt2, Zip1-4 and Irt1, have recently been characterized. Zrt1, Zrt2 and Zip1-4 are probably zinc transporters in Saccharomyces cerevisiae and Arabidopsis thaliana whereas Irt1 appears to play a role in iron uptake in A. thaliana. The family of proteins including these functionally characterized transporters has been designated the Zrt- and Irt-related protein (ZIP) family. In this report, ZIP family proteins in the current databases were identified and multiply aligned, and a phylogenetic tree for the family was constructed. A family specific signature sequence was derived, and the available sequences were analyzed for residues of potential functional significance. A fully conserved intramembranous histidyl residue, present within a putative amphipathic, alpha-helical, transmembrane spanning segment, was identified which may serve as a part of an intrachannel heavy metal ion binding site. The occurrence of a proposed extramembranal metal binding motif (H X H X H) was examined in order to evaluate its potential functional significance for various members of the family. The computational analyses reported in this topical review should serve as a guide to future researchers interested in the structure-function relationships of ZIP family proteins.

Amino Acid Sequence↗