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Huimin Zhao

Publications and source records attributed to Huimin Zhao.

At least 37 records · Page 2Linked to original sources
PubMed2006

Probing the substrate specificity of aminopyrrolnitrin oxygenase (PrnD) by mutational analysis.

Molecular modeling and mutational analysis (site-directed mutagenesis and saturation mutagenesis) were used to probe the molecular determinants of the substrate specificity of aminopyrrolnitrin oxygenase (PrnD) from Pseudomonas fluorescens Pf-5. There are 17 putative substrate-contacting residues, and mutations at two of the positions, positions 312 and 277, could modulate the enzyme substrate specificity separately or in combination. Interestingly, several of the mutants obtained exhibited higher catalytic efficiency (approximately two- to sevenfold higher) with the physiological substrate aminopyrrolnitrin than the wild-type enzyme exhibited.

Amines↗
PubMed2006

Recent advances in biocatalysis by directed enzyme evolution.

Naturally occurring enzymes are remarkable biocatalysts with numerous potential applications in industry and medicine. However, many of their catalyst properties often need to be further tailored to meet the specific requirements of a given application. Within this context, directed evolution has emerged over the past decade as a powerful tool for engineering enzymes with new or improved functions. This review summarizes recent advances in applying directed evolution approaches to alter various enzyme properties such as activity, selectivity (enantio- and regio-), substrate specificity, stability, and solubility. Special attention will be paid to the creation of novel enzyme activities and products by directed evolution.

Bacteria↗
PubMed2006

Optimizing a biocatalyst for improved NAD(P)H regeneration: directed evolution of phosphite dehydrogenase.

Cofactor regeneration is an important solution to the problem of implementing complex cofactor requiring enzymatic reactions at the industrial scale. NAD(P)H-dependent oxidoreductases are highly valuable biocatalysts, but the high cost of the nicotinamide cofactors necessitates in situ cofactor regeneration for preparative applications. Here we report the use of directed evolution to enhance the industrially important properties of phosphite dehydrogenase for NAD(P)H regeneration. A two-tiered sorting method of selection and screening was used in conjunction with random and rational mutagenesis. Following six rounds of directed evolution, soluble expression in E. coli was increased more than 3-fold, while the turnover rate was increased about 2-fold, effectively lowering the cost of the enzyme by >6-fold. Large-scale production of the final mutant enzyme by fermentation resulted in approximately 6-times higher yield (Units/Liter) than the WT enzyme. The enhancements of PTDH were independent of expression vector and E. coli strain utilized. The advantage of the final mutant over the WT enzyme was demonstrated using the industrially relevant bioconversion of trimethylpyruvate to L-tert-leucine. The mutations discovered are discussed in the context of a three dimensional structural model and the resulting changes in kinetics and soluble expression. The engineered phosphite dehydrogenase has great potential for NAD(P)H regeneration in industrial biocatalysis.

Catalysis↗
PubMed2006

[Preparation and physicochemical properties for zirconia as high performance liquid chromatographic packing materials].

There has been an increasing importance in the application of zirconia as column packing material for high performance liquid chromatography (HPLC) because of its excellent chemical, thermal, and mechanical stabilities as well as bioanalysis suitability. Zirconia spheres could be synthesized by polymerization-induced colloid aggregation (PICA), oil emulsion methods (OEM), or spray-dry method. It has been found that preparative procedure of zirconia support can affect its chromatographic behavior significantly. The review analyzes the relationship between zirconia packings' physicochemical properties and their preparative procedures. Specific surface area, pore structure, pore volume, pore size distribution, and particle size distribution are discussed in detail. At present, zirconia has not become highly competitive because of its limitations such as small specific surface area and pore volume, broad pore size distribution and particle distribution, and unfavorable pore structure. The review also describes silica-zirconia composite HPLC materials prepared by sol-gel method, coating and co-precipitation procedure. However, their chemical stability as HPLC column packings was suspicious because of the residual silica on the material surface. Furthermore, the study showed that zircon (ZrSiO4) is present in all silica-coated zirconia or silica-zirconia composites. It could be deduced that co-existence of more than one crystallines on the material surface could produce chemical heterogeneity, resulting in complex chromatographic separation mechanism. It leads a conclusion that a new synthetic way is needed for preparing zirconia-based HPLC packings with ideal chromatographic parameters. A novel material with higher specific surface area and pore volume, good pore structure, narrower pore size distribution and more uniform particle size distribution was prepared by layer-by-layer self-assembly technique (LbL SA), consisting of micrometer-sized silica spheres as core and nanometer-size zirconia particles as surface coating. The chemical stability of the material is as good as that of zirconia, and its specific surface area is as high as that of silica.

English Abstract↗
PubMed2005

A highly sensitive selection method for directed evolution of homing endonucleases.

Homing endonucleases are enzymes that catalyze DNA sequence specific double-strand breaks and can significantly stimulate homologous recombination at these breaks. These enzymes have great potential for applications such as gene correction in gene therapy or gene alteration in systems biology and metabolic engineering. However, homing endonucleases have a limited natural repertoire of target sequences, which severely hamper their applications. Here we report the development of a highly sensitive selection method for the directed evolution of homing endonucleases that couples enzymatic DNA cleavage with the survival of host cells. Using I-SceI as a model homing endonuclease, we have demonstrated that cells with wild-type I-SceI showed a high cell survival rate of 80-100% in the presence of the original I-SceI recognition site, whereas cells without I-SceI showed a survival rate <0.003%. This system should also be readily applicable for directed evolution of other DNA cleavage enzymes.

Deoxyribonucleases, Type II Site-Specific↗
PubMed2005

Reconstitution and characterization of aminopyrrolnitrin oxygenase, a Rieske N-oxygenase that catalyzes unusual arylamine oxidation.

Rieske oxygenases catalyze a wide variety of important oxidation reactions. Here we report the characterization of a novel Rieske N-oxygenase, aminopyrrolnitrin oxygenase (PrnD) that catalyzes the unusual oxidation of an arylamine to an arylnitro group. PrnD from Pseudomonas fluorescens Pf5 was functionally expressed in Escherichia coli, and the activity of the purified PrnD was reconstituted, which required in vitro assembly of the Rieske iron-sulfur cluster into the protein and the presence of NADPH, FMN, and an E. coli flavin reductase SsuE. Biochemical and bioinformatics studies indicated that the reconstituted PrnD contains a Rieske iron-sulfur cluster and a mononuclear iron center that are formed by residues Cys(69), Cys(88), His(71), His(91), Asp(323), His(186), and His(191), respectively. The enzyme showed a limited range of substrate specificity and catalyzed the conversion of aminopyrrolnitrin into pyrrolnitrin with K(m) = 191 microM and k(cat) = 6.8 min(-1). Isotope labeling experiments with (18)O(2) and H(2)(18)O suggested that the oxygen atoms in the pyrrolnitrin product are derived exclusively from molecular oxygen. In addition, it was found that the oxygenation of the arylamine substrates catalyzed by PrnD occurs at the enzyme active site and does not involve free radical chain reactions. By analogy to known examples of arylamine oxidation, a catalytic mechanism for the bioconversion of amino pyrrolnitrin into pyrrolnitrin was proposed. Our results should facilitate further mechanistic and crystallographic studies of this arylamine oxygenase and may provide a new enzymatic route for the synthesis of aromatic nitro compounds from their corresponding aromatic amines.

Amines↗
PubMed2005

Biosynthesis of phloroglucinol.

Substantial concentrations of phloroglucinol were synthesized by Pseudomonas fluorescens Pf-5 expressing the plasmid-localized phlACBDE gene cluster responsible for biosynthesis of 2,4-diacetylphloroglucinol. Expression in Escherichia coli of a single gene in this cluster, P. fluorescens Pf-5 phlD, led to extracellular accumulation of phloroglucinol. Purification of PhlD to homogeneity afforded an enzyme that catalyzed the conversion of malonyl-CoA into phloroglucinol with Km = 5.6 muM and kcat = 10 min-1. Acetylase and deacetylase activities were observed with the catalyzed interconversions of phloroglucinol, 2-acetylphloroglucinol, and 2,4-diacetylphloroglucinol when phlACB was expressed in E. coli. Beyond the mechanistic implications attendant with the identification of an enzyme that catalyzes the conversion of malonyl-CoA into phloroglucinol, PhlD provides the basis for environmentally benign syntheses of phloroglucinol and resorcinol from glucose.

Bacterial Proteins↗
PubMed2005

Enhancement of p,p'-DDT photodegradation on soil surfaces using TiO2 induced by UV-light.

Enhancement of p,p'-DDT photodegradation on soil surfaces using TiO2 induced by UV-light was mainly investigated in this work. After being spiked with p,p'-DDT, soil samples loaded with different doses of TiO2 (0%, 0.5%, 1%, 2%, and 3% wt) were exposed to UV-light irradiation for 24 h. The results indicated that the photodegradation of p,p'-DDT followed the pseudo-first-order kinetics. TiO2 accelerated the photodegradation of p,p'-DDT significantly as indicated by the half-life reduction from 23.3 h to 10.4 h, corresponding to the TiO2 content from 0% to 3% respectively. In addition, the effects of soil pH, photon flux and humic substances on p,p'-DDT degradation were investigated. The photodegradation rate increased with the increase of the soil pH and photon flux. The humic substances (2% wt) inhibited the p,p'-DDT photodegradation by reducing the amount of light available to excite the p,p'-DDT and TiO2 or by quenching radicals capable of oxidizing p,p'-DDT. p,p'-DDE, p,p'-DDD and DDMU were main degradation intermediates and they were further degraded in the presence of TiO2.

Biodegradation, Environmental↗
PubMed2005

Directed evolution of specific receptor-ligand pairs for use in the creation of gene switches.

Despite their versatility and power in controlling gene regulation in nature, nuclear hormone receptors (NHRs) have largely eluded utility in heterologous gene regulation applications such as gene therapy and metabolic engineering. The main reason for this void is the pleiotropic interference of the receptor-ligand combination with regulatory networks in the host organism. In recent years, numerous strategies have been developed to engineer ligand-receptor pairs that do not cross-interact with host regulatory pathways. However, these strategies have either met with limited success or cannot be readily extended to other ligand-receptor pairs. Here, we present a simple, effective, and readily generalizable strategy for reengineering NHRs to respond specifically to a selected synthetic ligand. The method involves generation of genetic diversity by stepwise individual site saturation mutagenesis of a fixed set of ligand-contacting residues and random point mutagenesis, followed by phenotypic screening based on a yeast two-hybrid system. As a test case, this method was used to alter the specificity of the NHR human estrogen receptor alpha in favor of the synthetic ligand 4,4'-dihydroxybenzil, relative to the natural ligand 17beta-estradiol, by >10(7)-fold. The resulting ligand-receptor pair is highly sensitive to the synthetic ligand in human endometrial cancer cells and is essentially fully orthogonal to the wild-type receptor-natural ligand pair. This method should provide a powerful, broadly applicable tool for engineering receptors/enzymes with improved or novel ligand/substrate specificity.

Binding Sites↗
PubMed2005

Rapid creation of a novel protein function by in vitro coevolution.

We have developed a simple and efficient method for creation of novel protein functions in an existing protein scaffold. The in vitro coevolution method involves design of a hypothetical pathway for the target function followed by stepwise directed evolution of the corresponding protein along the pathway. As a test case, this strategy was used to engineer variants of human estrogen receptor alpha ligand-binding domain (hERalphaLBD) with novel corticosterone activity. Two steroids, testosterone and progesterone, that provide a progressive structural bridge between 17beta-estradiol and corticosterone, were chosen to assist the directed evolution of hERalphaLBD. A total of approximately 10(6) variants were screened in four rounds of random mutagenesis, resulting in two hERalphaLBD variants that respond to corticosterone. Creation of this new ligand activity required the presence of four simultaneous mutations. In addition, several required mutations were located outside the ligand binding pocket and yet exerted important action on ligand binding. Our results demonstrate the ability of in vitro coevolution to create novel protein function that is difficult or impossible to achieve by existing protein engineering approaches and also shed light on the natural evolution of nuclear hormone receptors. This in vitro coevolution approach should provide a powerful, broadly applicable tool for engineering biological molecules and systems with novel functions.

Corticosterone↗
PubMed2005

Creating new specific ligand-receptor pairs for transgene regulation.

The creation of specifically matched ligand-receptor pairs that are orthogonal to naturally present interacting pairs is essential for the development of small molecule-regulated gene expression systems for biotechnological applications. However, for many years this task has represented a significant challenge for synthetic chemists and protein engineers. Recently, Doyle and colleagues demonstrated that highly specific ligand-receptor pairs can be engineered in a rapid fashion by creating large libraries of protein variants and applying a selection scheme to identify variants with improved activation by the target synthetic ligand.

Binding Sites↗
PubMed2005

Mechanistic investigation of a highly active phosphite dehydrogenase mutant and its application for NADPH regeneration.

NAD(P)H regeneration is important for biocatalytic reactions that require these costly cofactors. A mutant phosphite dehydrogenase (PTDH-E175A/A176R) that utilizes both NAD and NADP efficiently is a very promising system for NAD(P)H regeneration. In this work, both the kinetic mechanism and practical application of PTDH-E175A/A176R were investigated for better understanding of the enzyme and to provide a basis for future optimization. Kinetic isotope effect studies with PTDH-E175A/A176R showed that the hydride transfer step is (partially) rate determining with both NAD and NADP giving (D)V values of 2.2 and 1.7, respectively, and (D)V/K(m,phosphite) values of 1.9 and 1.7, respectively. To better comprehend the relaxed cofactor specificity, the cofactor dissociation constants were determined utilizing tryptophan intrinsic fluorescence quenching. The dissociation constants of NAD and NADP with PTDH-E175A/A176R were 53 and 1.9 microm, respectively, while those of the products NADH and NADPH were 17.4 and 1.22 microm, respectively. Using sulfite as a substrate mimic, the binding order was established, with the cofactor binding first and sulfite binding second. The low dissociation constant for the cofactor product NADPH combined with the reduced values for (D)V and k(cat) implies that product release may become partially rate determining. However, product inhibition does not prevent efficient in situ NADPH regeneration by PTDH-E175A/A176R in a model system in which xylose was converted into xylitol by NADP-dependent xylose reductase. The in situ regeneration proceeded at a rate approximately fourfold faster with PTDH-E175A/A176R than with either WT PTDH or a NADP-specific Pseudomonas sp.101 formate dehydrogenase mutant with a total turnover number for NADPH of 2500.

Deuterium↗
PubMed2005

Directed evolution of a thermostable phosphite dehydrogenase for NAD(P)H regeneration.

NAD(P)H-dependent oxidoreductases are valuable tools for synthesis of chiral compounds. The expense of the cofactors, however, requires in situ cofactor regeneration for preparative applications. We have attempted to develop an enzymatic system based on phosphite dehydrogenase (PTDH) from Pseudomonas stutzeri to regenerate the reduced nicotinamide cofactors NADH and NADPH. Here we report the use of directed evolution to address one of the main limitations with the wild-type PTDH enzyme, its low stability. After three rounds of random mutagenesis and high-throughput screening, 12 thermostabilizing amino acid substitutions were identified. These 12 mutations were combined by site-directed mutagenesis, resulting in a mutant whose T50 is 20 degrees C higher and half-life of thermal inactivation at 45 degrees C is >7,000-fold greater than that of the parent PTDH. The engineered PTDH has a half-life at 50 degrees C that is 2.4-fold greater than the Candida boidinii formate dehydrogenase, an enzyme widely used for NADH regeneration. In addition, its catalytic efficiency is slightly higher than that of the parent PTDH. Various mechanisms of thermostabilization were identified using molecular modeling. The improved stability and effectiveness of the final mutant were shown using the industrially important bioconversion of trimethylpyruvate to l-tert-leucine. The engineered PTDH will be useful in NAD(P)H regeneration for industrial biocatalysis.

Amino Acid Substitution↗
PubMed2004

Directed evolution of soluble single-chain human class II MHC molecules.

Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers that present antigenic peptides to T cells. Expression of these molecules in soluble form has met limited success, presumably due to their large size, heterodimeric structure and the presence of multiple disulfide bonds. Here we have used directed evolution and yeast surface display to engineer soluble single-chain human lymphocyte antigen (HLA) class II MHC DR1 molecules without covalently attached peptides (scDR1alphabeta). Specifically, a library of mutant scDR1alphabeta molecules was generated by random mutagenesis and screened by fluorescence activated cell sorting (FACS) with DR-specific conformation-sensitive antibodies, yielding three well-expressed and properly folded scDR1alphabeta variants displayed on the yeast cell surface. Detailed analysis of these evolved variants and a few site-directed mutants generated de novo indicated three amino acid residues in the beta1 domain are important for the improved protein folding yield. Further, molecular modeling studies suggested these mutations might increase the protein folding efficiency by improving the packing of a hydrophobic core in the alpha1beta1 domain of DR1. The scDR1alphabeta mutants displayed on the yeast cell surface are remarkably stable and bind specifically to DR-specific peptide HA(306-318) with high sensitivity and rapid kinetics in flow cytometric assays. Moreover, since the expression, stability and peptide-binding properties of these mutants can be directly assayed on the yeast cell surface using immuno-fluorescence labeling and flow cytometry, time-consuming purification and refolding steps of recombinant DR1 molecules are eliminated. Therefore, these scDR1alphabeta molecules will provide a powerful technology platform for further design of DR1 molecules with improved peptide-binding specificity and affinity for therapeutic and diagnostic applications. The methods described here should be generally applicable to other class II MHC molecules and also class I MHC molecules for their functional expression, characterization and engineering.

Directed Molecular Evolution↗
PubMed2004

Directed evolution of human estrogen receptor variants with significantly enhanced androgen specificity and affinity.

Human estrogen receptor alpha (hERalpha) and human androgen receptor exhibit exquisite ligand specificity, which underlies their remarkable ability to effect ligand-regulated gene transcription in a highly distinctive and specific manner. Here we used a directed evolution approach to create hERalpha variants with enhanced androgen specificity and affinity with the goal to better understand the molecular basis of ER ligand specificity and the evolutionary mechanism of nuclear receptors. We developed a sensitive yeast two-hybrid system to screen for hERalpha variants with increased transactivation potency toward testosterone. After two rounds of directed evolution, we identified five hERalpha variants with dramatically improved transactivation potency toward testosterone in both yeast and mammalian cells. These variants showed up to 7,600-fold improvement in the binding affinity for testosterone and only slightly reduced affinity toward 17beta-estradiol. Detailed analysis of these evolved variants and a few site-directed mutants generated de novo led to several unexpected findings including the following. 1) Only two beneficial mutations were needed to create hERalpha variants with near nanomolar affinity for testosterone. 2) Some beneficial mutations were synergistic, context-dependent, or non-additive. 3) Of the five identified beneficial mutations, four of them were not in the ER ligand binding pocket and yet exerted important action on ligand specificity. 4) The single ligand-contacting mutation E353Q plays a dominant role in discriminating androgens and estrogens. These results, viewed in conjunction with the ligand exploitation model of nuclear receptor evolution, suggest that the mutation E353Q may represent a key event in the evolution of androgen receptors from an ancestral estrogen receptor and that ligand promiscuity may play an important role in the creation of new nuclear receptors via divergent evolution.

Amino Acid Sequence↗
PubMed2004

Rational pathway engineering of type I fatty acid synthase allows the biosynthesis of triacetic acid lactone from D-glucose in vivo.

Metabolic pathway engineering is a powerful tool to synthesize structurally diverse and complex chemicals via genetic manipulation of multistep catalytic systems involved in cell metabolism. Here, we report the rational design of a fatty acid biosynthetic pathway, Brevibacterium ammoniagenes fatty acid synthase B (FAS-B), that allows the microbial synthesis of triacetic acid lactone (TAL) from an inexpensive feedstock, d-glucose. TAL can be chemically converted to phloroglucinol, which is a core structure for the synthesis of various high value bioactive compounds and energetic compounds such as 1,3,5-triamino-2,4,6-trinitrobenzene (TATB). Synthesis of phloroglucinol from d-glucose using this combined biological and chemical synthesis may offer significant advantages over the current phloroglucinol manufacture, including environmental friendliness and reduction in the cost of phloroglucinol. More importantly, it represents a novel strategy for the benzene-free synthesis of aromatic chemicals.

Bacterial Proteins↗