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PubMed1984

Enzyme studies of replication of the Escherichia coli chromosome.

Progress of the replication forks of the Escherichia coli chromosome depends on a multisubunit DNA polymerase (for chain elongation) and a primosome (for chain initiations), together comprising about 30 polypeptides with a mass in excess of 10(6) daltons. Integration of their actions with those of helicases and DNA binding proteins suggest a more complex and integrated replisome assembly with novel possibilities for concurrent replication of both parental strands. Initiation of a new cycle of chromosome replication at its unique 245-bp (oriC) is being studied in a soluble enzyme system with plasmids, autonomous replication of which depends on the oriC sequence. Required proteins include RNA polymerase, DNA gyrase, dnaA protein (with 4 strong binding sites in oriC), HU protein, and additional proteins (e.g., topoisomerase I and ribonuclease H) that confer oriC specificity by suppressing initiation of replication elsewhere on the duplex DNA. Clarification of the biochemical mechanisms of replication is fundamental for understanding cell growth and development. Knowledge of the biochemistry of initiating a cycle of chromosome replication opens the way toward exploring the regulation of the cell cycle. I remain faithful to the conviction that anything a cell can do, a biochemist should be able to do. He should do it even better, being freed from the constraints of substrate and enzyme concentrations, pH, ionic strength, and temperature, and by having the license to introduce novel reagents to drive or restrain a reaction. Put another way, one can be creative more easily with a reconstituted system. One can grapple directly with the molecules instead of trying by remote means to manipulate their structures or levels in the intact cell. Enzyme purification carries many dangers beyond the well-known exposure of the fragile enzyme to the hostilities of an unfamiliar environment, high dilution, glass containers and a denaturable investigator. But the rewards of enzyme purification have justified the effort. The polymerases, nucleases, ligases purified out of curiosity about the mechanisms of replication, repair and recombination have supplied the cast of actors responsible for the current drama of genetic engineering. Beyond the uses of these enzymes as reagents, understanding the mechanisms of DNA metabolism will have practical value in manipulating the replication of plasmids and viruses and the expression of their genes and, beyond that, in obtaining a more secure grasp of chromosome structure and function.

Bacterial Proteins↗
PubMed2005

Structure-based chemical modification strategy for enzyme replacement treatment of phenylketonuria.

Structure-based protein engineering coupled with chemical modifications (e.g., pegylation) is a powerful combination to significantly improve the development of proteins as therapeutic agents. As a test case, phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) was selected for enzyme replacement therapy in phenylketonuria [C.R. Scriver, S. Kaufman, Hyperphenylalaninemia:phenylalanine Hydroxylase Deficiency. The Metabolic and Molecular Bases of Inherited Disease, McGraw-Hill, New York, 2001, Chapter 77], an inherited metabolic disorder (OMIM 261600) causing mental retardation due to deficiency of the enzyme l-phenylalanine hydroxylase (EC 1.14.16.1). Previous in vivo studies of recombinant PAL demonstrated a lowering of blood l-phenylalanine levels; yet, the metabolic effect was not sustained due to protein degradation and immunogenicity [C.N. Sarkissian, Z. Shao, F. Blain, R. Peevers, H. Su, R. Heft, T.M. Chang, C.R. Scriver, A different approach to treatment of phenylketonuria:phenylalanine degradation with recombinant phenylalanine ammonia lyase, Proc. Natl. Acad. Sci. USA 96 (1999) 2339; J.A. Hoskins, G. Jack, H.E. Wade, R.J. Peiris, E.C. Wright, D.J. Starr, J. Stern, Enzymatic control of phenylalanine intake in phenylketonuria, Lancet 1 (1980) 392; C.M. Ambrus, S. Anthone, C. Horvath, K. Kalghatgi, A.S. Lele, G. Eapen, J.L. Ambrus, A.J. Ryan, P. Li, Extracorporeal enzyme reactors for depletion of phenylalanine in phenylketonuria, Ann. Intern. Med. 106 (1987) 531]. Here, we report the 1.6A three-dimensional structure of Rhodosporidium toruloides PAL, structure-based molecular engineering, pegylation of PAL, as well as in vitro and in vivo PKU mouse model studies on pegylated PAL formulations. Our results show that pegylation of R. toruloides PAL leads to promising therapeutic efficacy after subcutaneous injection by enhancing the in vivo activity, lowering plasma phenylalanine, and leading to reduced immunogenicity. The three-dimensional structure of PAL provides a basis for understanding the properties of pegylated forms of PAL and strategies for structure-based re-engineering of PAL for PKU treatment.

Animals↗
PubMed2001

DNA polymerases as engines for biotechnology.

This review examines the DNA polymerases, a class of enzymes that has been an essential tool for molecular biology research. Several families of DNA polymerases have been defined based on amino acid sequence comparisons, and new enzymes are continually being discovered, driving the expansion of the current classifications. Structural similarities among the enzymes are examined, as well as the functions of the various subunits and enzyme domains. The natural variety of polymerase activities has been harnessed for applications such as amplification, labeling, and detection of DNA sequences. In addition, enhancements to DNA polymerases by genetic engineering will be described, such as enzymes specifically designed for DNA sequencing by improving the incorporation of dideoxynucleotide terminators. Reverse transcription, the ability to use RNA as a template for DNA synthesis, is described for the application of making cDNA. We believe that new and unanticipated applications will emerge as new polymerases and mutated polymerases are created and characterized.

Biotechnology↗
PubMed1998

Exchange of nucleoside monophosphate-binding domains in adenylate kinase and UMP/CMP kinase.

Two types of active chimeric enzymes have been constructed by genetic engineering of chicken cytosolic adenylate kinase (AK) and porcine brain UMP/CMP kinase (UCK): one, designated as UAU, carries an AMP-binding domain of AK in the remaining body of UCK; and the other, designated as AUA, carries a UMP/CMP-binding domain of UCK in the remaining body of AK. Steady-state kinetic analysis of these chimeric enzymes revealed that UAU is 4-fold more active for AMP, 40-fold less active for UMP, and 4-fold less active for CMP than the parental UCK, although AUA has considerably lowered reactivity for both AMP and UMP. Circular dichroism spectra of the two chimeric enzymes suggest that UAU and AUA have similar folding structures to UCK and AK, respectively. Furthermore, proton NMR measurements of the UCK and UAU proteins indicate that significant differences in proton signals are limited to the aromatic region, where an imidazole C2H signal assigned to His31 shows a downfield shift upon conversion of UCK to UAU, and the signals assigned to Tyr49 and Tyr56 in the UMP/CMP-binding domain disappear in UAU. In contrast, AUA has a Tm value about 11 degreesC lower than AK, whereas UAU and UCK have similar Tm values. These results together show that the substrate specificity of nucleoside monophosphate (NMP) kinases can be engineered by the domain exchange, even though the base moiety of NMP appears to be recognized cooperatively by both the NMP-binding domain and the MgATP-binding core domain.

Adenylate Kinase↗
PubMed1991

Current status of biotechnology in China.

There has been increasing interest over past decade in exploring the possibility of using new biotechnology to produce new products and to improve the old productive process. The researches and applications of genetic engineering, cell fusion, mutagenesis, cell and enzyme immobilization in enzyme, antibiotic, vitamins, steroid, amino acid, organic acid, solvent, food and brewage industries is reviewed.

Anti-Bacterial Agents↗
PubMed1994

Engineering of a metal coordinating site into human glutathione transferase M1-1 based on immobilized metal ion affinity chromatography of homologous rat enzymes.

Rat glutathione transferase (GST) 3-3 binds to Ni(II)-iminodiacetic acid (IDA)-agarose, whereas other GSTs that are abundant in rat liver do not bind to this immobilized metal ion affinity chromatography (IMAC) adsorbent. Rat GST 3-3 contains two superficially located amino acid residues, His84 and His85, that are suitably positioned for coordination to Ni(II)-IDA-agarose. This particular structural motif is lacking in GSTs that do not bind to the IMAC matrix. Creation of an equivalent His-His structure in the homologous human GST M1-1 by protein engineering afforded a mutant enzyme that displays affinity for Ni(II)-IDA-agarose, in contrast to the wild-type GST M1-1. The results identify a distinct site that is operational in IMAC and suggest an approach to the rational design of novel integral metal coordination sites in proteins.

Amino Acid Sequence↗
PubMed1996

The mechanism of velocity modulated allosteric regulation in D-3-phosphoglycerate dehydrogenase. Cross-linking adjacent regulatory domains with engineered disulfides mimics effector binding.

D-3-Phosphoglycerate dehydrogenase (PGDH) (EC 1.1.1.95) from Escherichia coli is an allosterically regulated enzyme of the Vmax type. It is a tetramer of identical subunits and each subunit is made up of three identifiable domains, the cofactor binding domain, the substrate binding domain, and the regulatory domain. Each subunit contacts two other subunits through adjacent cofactor binding domains and through adjacent regulatory domains. L-Serine, the physiological effector, inhibits catalytic activity by apparently tethering regulatory domains from adjacent subunits together through the formation of hydrogen bonds to each subunit. This investigation demonstrates that cross-linking adjacent regulatory domains with engineered disulfides produces catalytic inhibition in the absence of inhibitor in a manner similar to that produced by the inhibitor. The inhibition due to cross-linking can be completely reversed in a concentration dependent manner by dithiothreitol. The active mutant enzyme, containing the engineered cysteines in the reduced state, retains its ability to be inhibited by L-serine, although at a 100-fold higher concentration. Hill plots of the serine inhibition of mutant and native enzyme indicate that the number of interacting sites remains at 2 in the mutant enzyme. The reversible inhibition of enzyme activity that results from tethering adjacent regulatory domains with engineered disulfides suggests that these domains move in some manner relative to one another during the active to inhibited state transition. These observations support the model which predicts that catalytic activity is regulated by the movement of rigid domains about flexible hinges and that effector binding prevents this by locking the regulatory domains in a state that produces an open active site cleft.

Allosteric Regulation↗
PubMed2000

Heterologous expression of xenobiotic mammalian-metabolizing enzymes in mutagenicity tester bacteria: an update and practical considerations.

There is an increasing need for metabolic competent cell systems for the mechanistic studies of biotransformation of xenobiotics in toxicology in general and in genotoxicology in particular. These cell systems combine the heterologous expression of a particular mammalian biotransformation enzyme with a specific target/ end point by which a functional analysis of the expressed gene product in the (geno)toxicity of chemicals can be performed. cDNAs of an increasing number of mammalian biotransformation enzymes is being cloned. The construction of specific expression vectors permits their heterologous expression in laboratory bacteria, such as Escherichia coli strains. This development does not only allow biochemical and enzymatic studies of (pure) enzyme preparations but also facilitates the engineering of metabolically competent mutagenicity tester bacteria, thereby providing new tools for genotoxicity testing and for studying of the roles of biotransformation in chemical carcinogenesis. In this review, we describe an update as well as an evaluation of enzymes expressed in mutagenicity tester bacteria. Four types of biotransformation enzymes are now expressed in these bacteria, namely, GSTs, CYPs, NATs, and STs. The expression of these enzymes in the tester bacteria and their subsequent application in mutagenicity assays demonstrates that heterologous expression in this type of bacteria has a number implications for the functionality of the biotransformation enzymes as well as for the functioning of the tester bacteria in mutagenicity detection. We also describe here a number of practical considerations in this regard.

Animals↗
PubMed1992

Deletion of the zinc-binding motif of CD13/aminopeptidase N molecules results in loss of epitopes that mediate binding of inhibitory antibodies.

The myeloid cell-surface glycoprotein CD13/aminopeptidase N (APN; EC 3.4.11.2) contains a pentapeptide (HExxH) in its extracellular domain that is characteristic of many zinc-dependent metalloproteinases. This region contains residues important for zinc binding and constitutes part of the catalytic domain of several metalloproteases. We deleted an internal fragment of 117 base pairs (bp) from the human CD13/APN cDNA, resulting in an in-frame deletion that included the sequences coding for this pentapeptide motif. The mutant cDNA was subcloned into a retroviral expression vector, and polypeptides encoded by the altered cDNA were expressed in transfected murine NIH-3T3 fibroblasts. The mutant CD13/APN molecules lacked enzymatic activity, and their intracellular processing to the cell surface was retarded by comparison with normal CD13/APN polypeptides. The mutant molecules also lacked epitopes required for binding of four of 19 CD13-specific monoclonal antibodies (MoAbs) tested in flow cytometric assays. Each of the four MoAbs also inhibited the enzymatic activity of wild-type APN molecules, suggesting that these antibodies may inhibit aminopeptidase activity by interfering with the enzyme's zinc-coordinating properties. Cells engineered to express mutant CD13/APN polypeptides at the cell surface provide a tool for defining the physiologic role of this enzyme on normal and malignant myeloid cells and marrow stromal cells.

Aminopeptidases↗
PubMed2026

Engineered MXene-based nanozyme platform: NIR-II photothermal and dual enzyme-mimetic potentiated chemodynamic synergy for precision tumor eradication.

The antioxidant defense barrier in the tumor microenvironment, particularly glutathione (GSH), considerably restricts the therapeutic efficacy of chemodynamic therapy (CDT). Moreover, CDT generally exhibits relatively mild therapeutic efficacy owing to its intrinsic reaction kinetics, making it difficult to achieve complete tumor eradication within a short time. To address these issues, we construct a functionalized nanotherapeutic platform, Nb2CTx@Ru-PEG2000-FA (NCRPF), for tumor photothermal ablation and enhanced CDT resulting from GSH depletion. NCRPF possesses three key advantages: 1. Efficient near-infrared II photothermal conversion (η = 42.08%), raising the tumor temperature above 45 °C within 90 s for rapid ablation; 2. Dual peroxidase-like and glutathione peroxidase-like activities, simultaneously depleting GSH and generating a burst of ·OH to eliminate residual tumors; 3. Targeted tumor accumulation with 2.9-fold higher efficiency than passive diffusion. Both in vitro and in vivo results confirm that this combined strategy achieves complete tumor eradication with favorable biosafety. Collectively, the NCRPF nanotherapeutic system provides a powerful new paradigm with high translational potential for the complete eradication of breast cancer.

Animals↗
PubMedDate not supplied

Construction of a cultivation system of a yeast single cell in a cell chip microchamber.

A novel single cell screening system was constructed using a yeast cell chip in combination with the yeast cell surface engineering [NanoBiotechnology 2005, 1, 105-111]. Enzymes or functional proteins displayed on a yeast cell surface can be used as a protein cluster. To achieve high-throughput screening of protein libraries on the cell surface, a catalytic reaction by a single cell-surface-engineered yeast cell was successfully carried out in the microchamber on the yeast cell chip. After screening, to replicate a target cell for use in measuring of activity, DNA sequencing, and preservation, a novel single cell cultivation system in the yeast cell chip was constructed. To avoid damage of the rapid dry up of medium in the microchamber array, the yeast cell chip was modified with a protection sheet, so that the modified chip was like a micro-culture tank constructed on the yeast cell chip microchamber. As a result, single yeast cell cultivation in the yeast cell chip microchamber was observed, and the modified yeast cell chip was evaluated to be good for a single cell selection. The improvement showed that the single cell screening system coupled with the single cell cultivation using the modified yeast cell chip may be superior to that by a cell sorter for the isolation of a target cell and its practical use.

Cell Count↗
PubMed2006

Protein folding and the robustness of cells.

The intricate intracellular infrastructure of all known life forms is based on proteins. The folded shape of a protein determines both the protein's function and the set of molecules it will bind to. This tight coupling between a protein's function and its interconnections in the molecular interaction network has consequences for the molecular course of evolution. It is also counter to human engineering approaches. Here we report on a simulation study investigating the impact of random errors in an abstract metabolic network of 500 enzymes. Tight coupling between function and interconnectivity of nodes is compared to the case where these two properties are independent. Our results show that the model system under consideration is more robust if function and interconnection are intertwined. These findings are discussed in the context of nanosystems engineering.

Cells↗
PubMed1999

Extremozymes.

Extremozymes offer new opportunities for biocatalysis and biotransformations as a result of their extreme stability. From recent work, major approaches to extending the range of applications of extremozymes have emerged. Both the discovery of new extremophilic species and the determination of genome sequences provide a route to new enzymes, with the possibility that these will lead to novel applications. Of equal importance, protein engineering and directed evolution provide approaches to improve enzyme stability and modify specificity in ways that may not exist in the natural world.

Bacteria↗
PubMed2002

Crystal structure of the (R)-specific enoyl-CoA hydratase from Aeromonas caviae involved in polyhydroxyalkanoate biosynthesis.

The (R)-specific enoyl coenzyme A hydratase ((R)-hydratase) from Aeromonas caviae catalyzes the addition of a water molecule to trans-2-enoyl coenzyme A (CoA), with a chain-length of 4-6 carbons, to produce the corresponding (R)-3-hydroxyacyl-CoA. It forms a dimer of identical subunits with a molecular weight of about 14,000 and is involved in polyhydroxyalkanoate (PHA) biosynthesis. The crystal structure of the enzyme has been determined at 1.5-A resolution. The structure of the monomer consists of a five-stranded antiparallel beta-sheet and a central alpha-helix, folded into a so-called "hot dog" fold, with an overhanging segment. This overhang contains the conserved residues including the hydratase 2 motif residues. In dimeric form, two beta-sheets are associated to form an extended 10-stranded beta-sheet, and the overhangs obscure the putative active sites at the subunit interface. The active site is located deep within the substrate-binding tunnel, where Asp(31) and His(36) form a catalytic dyad. These residues are catalytically important as confirmed by site-directed mutagenesis and are possibly responsible for the activation of a water molecule and the protonation of a substrate molecule, respectively. Residues such as Leu(65) and Val(130) are situated at the bottom of the substrate-binding tunnel, defining the preference of the enzyme for the chain length of the substrate. These results provide target residues for protein engineering, which will enhance the significance of this enzyme in the production of novel PHA polymers. In addition, this study provides the first structural information of the (R)-hydratase family and may facilitate further functional studies for members of the family.

Aeromonas↗
PubMed2002

Developments and trends in enzyme catalysis in nonconventional media.

The conventional notion that enzymes are only active in aqueous media has long been discarded, thanks to the numerous studies documenting enzyme activities in nonaqueous media, including pure organic solvents and supercritical fluids. Enzymatic reactions in nonaqueous solvents offer new possibilities for producing useful chemicals (emulsifiers, surfactants, wax esters, chiral drug molecules, biopolymers, peptides and proteins, modified fats and oils, structured lipids and flavor esters). The use of enzymes in both macro- and microaqueous systems has been investigated especially intensively in the last two decades. Although enzymes exhibit considerable activity in nonaqueous media, the activity is low compared to that in water. This observation has led to numerous studies to modify enzymes for specific purposes by various means including protein engineering. This review covers the historical developments, major technological advances and recent trends of enzyme catalysis in nonconventional media. A brief description of different classes of enzymes and their use in industry is provided with representative examples. Recent trends including use of novel solvent systems, role of water activity, stability issues, medium and biocatalyst engineering aspects have been discussed with examples. Special attention is given to protein engineering and directed evolution.

Journal Article↗
PubMed1999

The crystal structure of the monomeric human SOD mutant F50E/G51E/E133Q at atomic resolution. The enzyme mechanism revisited.

The crystal structure of the engineered monomeric human Cu,ZnSOD triple mutant F50E/G51E/E133Q (Q133M2SOD) is reported at atomic resolution (1.02 A). This derivative has about 20 % of the wild-type activity. Crystals of Q133M2SOD have been obtained in the presence of CdCl2. The metal binding site is disordered, with both cadmium and copper ions simultaneously binding to the copper site. The cadmium (II) ions occupy about 45 % of the copper sites by binding the four histidine residues which ligate copper in the native enzyme, and two further water molecules to complete octahedral coordination. The copper ion is tri-coordinate, and the fourth histidine (His63) is detached from copper and bridges cadmium and zinc. X-ray absorption spectroscopy performed on the crystals suggests that the copper ion has undergone partial photoreduction upon exposure to the synchrotron light. The structure is also disordered in the disulfide bridge region of loop IV that is located at the subunit/subunit interface in the native SOD dimer. As a consequence, the catalytically relevant Arg143 residue is disordered. The present structure has been compared to other X-ray structures on various isoenzymes and to the solution structure of the same monomeric form. The structural results suggest that the low activity of monomeric SOD is due to the disorder in the conformation of the side-chain of Arg143 as well as of loop IV. It is proposed that the subunit-subunit interactions in the multimeric forms of the enzyme are needed to stabilize the correct geometry of the cavity and the optimal orientation of the charged residues in the active channel. Furthermore, the different coordination of cadmium and copper ions, contemporaneously present in the same site, are taken as models for the oxidized and reduced copper species, respectively. These properties of the structure have allowed us to revisit the enzymatic mechanism.

Binding Sites↗