Characterization of recombinant chloroperoxidase, and F103A and C29H/C79H/C87H mutants

Mechanistically and structurally chloroperoxidase (CPO) occupies a unique niche among heme containing enzymes. Chloroperoxidase catalyzes a broad range of reactions, such as oxidation of organic substrates, dismutation of hydrogen peroxide, and mono-oxygenation of organic molecules. To expand the synthetic utility of CPO and to appreciate the important interactions that lead to CPO’s exceptional properties, a site-directed mutagenesis study was undertaken. ^ Recombinant CPO and CPO mutants were heterologously expressed in Aspergillus niger. The overall protein structure was almost the same as that of wild type CPO, as determined by UV-vis, NMR and CD spectroscopies. Phenylalanine103, which was proposed to regulate substrate access to the active site by restricting the size of substrates and to control CPO’s enantioselectivity, was mutated to Ala. The ligand binding affinity and most importantly the catalytic activity of F103A was dramatically different from wild type CPO. The mutation essentially eliminated the chlorination and dismutation activities but enhanced, 4-10 fold, the epoxidation, peroxidation, and N-demethylation activities. As expected, the F103A mutant displayed dramatically improved epoxidation activity for larger, more branched styrene derivatives. Furthermore, F103A showed a distinctive enantioselectivity profile: losing enantioselectivity to styrene and cis-β-methylstyrene; having a different configuration preference on α-methylstyrene; showing higher enantioselectivites and conversion rates on larger, more branched substrates. Our results show that F103 acts as a switch box that controls the catalytic activity, substrate specificity, and product enantioselectivity of CPO. Given that no other mutant of CPO has displayed distinct properties, the results with F103A are dramatic. ^ The diverse catalytic activity of CPO has long been attributed to the presence of the proximal thiolate ligand. Surprisingly, a recent report on a C29H mutant suggested otherwise. A new CPO triple mutant C29H/C79H/C87H was prepared, in which all the cysteines were replaced by histidine to eliminate the possibility of cysteine coordinating to the heme. No active form protein was isolated, although, successful transformation and transcription was confirmed. The result suggests that Cys79 and Cys87 are critical to maintaining the structural scaffold of CPO. ^ In vitro biodegradation of nanotubes by CPO were examined by scanning electron microscope method, but little oxidation was observed. ^

Mechanisms of chloroperoxidase-catalyzed enantioselective reactions as probed by site-directed mutagenesis and isotopic labeling

Chloroperoxidase (CPO) is a heme-containing glycoprotein secreted by the marine fungus Caldariomyces fumago. Chloroperoxidase contains one ferriprotoporphyrin IX prosthetic group per molecule and catalyzes a variety of reactions, such as halogenation, peroxidation and epoxidation. The versatile catalytic activities of CPO coupled with the increasing demands for chiral synthesis have attracted an escalating interest in understanding the mechanistic and structural properties of this enzyme. In order to better understand the mechanisms of CPO-catalyzed enantioselective reactions and to fine-tune the catalytic properties of chloroperoxidase, asparagine 74 (N74) located in the narrow substrate access channel of CPO was replaced by a bulky, nonpolar valine and a polar glutamine using site-directed mutagenesis. The CPO N74 mutants displayed significantly enhanced activity toward nonpolar substrates compared to wild-type CPO as a result of changes in space and polarity of the heme distal environment. More interestingly, N74 mutants showed dramatically decreased chlorination and catalase activity but significantly enhanced epoxidation activity as a consequence of improved kinetic perfection introduced by the mutation as reflected by the favorable changes in k cat and kcat/KM of these reactions. It is also noted that the N74V mutant is capable of decomposing cyanide, the most notorious poison for many hemoproteins, as judged by the unique binding behavior of N74V with potassium cyanide. Histidine 105 (H105) was replaced by a nonpolar amino acid alanine using site-directed mutagenesis. The CPO H105 mutant (H105A) displayed dramatically decreased chlorination and catalase activity possibly because of the decreased polarity in the heme distal environment and loss of the hydrogen bonds between histidine 105 and glutamic acid 183. However, significantly increased enantioselectivity was observed for the epoxidation of bulky styrene derivatives. Furthermore, my study provides strong evidence for the proposed histidine/cysteine ligand switch in chloroperoxidase, providing experimental support for the structure of the 420-nm absorption maximum for a number of carbon monoxide complexes of heme-thiolate proteins. For the NMR study, [dCPO(heme)] was produced using 90% deuterated growth medium with excess heme precursors and [dCPO(Phe)] was grown in the same highly deuterated medium that had been supplemented with excess natural phenylalanine. To make complete heme proton assignments, NMR spectroscopy has been performed for high-resolution structural characterization of [dCPO(heme)] and [dCPO(Phe)] to achieve unambiguous and complete heme proton assignments, which also allows important amino acids close to the heme active center to be determined.

Mechanisms of Chloroperoxidase-catalyzed Enantioselective Reactions as Probed by Site-directed Mutagenesis and Isotopic Labeling

Chloroperoxidase (CPO) is a heme-containing glycoprotein secreted by the marine fungus Caldariomyces fumago. Chloroperoxidase contains one ferriprotoporphyrin IX prosthetic group per molecule and catalyzes a variety of reactions, such as halogenation, peroxidation and epoxidation. The versatile catalytic activities of CPO coupled with the increasing demands for chiral synthesis have attracted an escalating interest in understanding the mechanistic and structural properties of this enzyme. In order to better understand the mechanisms of CPO-catalyzed enantioselective reactions and to fine-tune the catalytic properties of chloroperoxidase, asparagine 74 (N74) located in the narrow substrate access channel of CPO was replaced by a bulky, nonpolar valine and a polar glutamine using site-directed mutagenesis. The CPO N74 mutants displayed significantly enhanced activity toward nonpolar substrates compared to wild-type CPO as a result of changes in space and polarity of the heme distal environment. More interestingly, N74 mutants showed dramatically decreased chlorination and catalase activity but significantly enhanced epoxidation activity as a consequence of improved kinetic perfection introduced by the mutation as reflected by the favorable changes in kcat and kcat/KM of these reactions. It is also noted that the N74V mutant is capable of decomposing cyanide, the most notorious poison for many hemoproteins, as judged by the unique binding behavior of N74V with potassium cyanide. Histidine 105 (H105) was replaced by a nonpolar amino acid alanine using site-directed mutagenesis. The CPO H105 mutant (H105A) displayed dramatically decreased chlorination and catalase activity possibly because of the decreased polarity in the heme distal environment and loss of the hydrogen bonds between histidine 105 and glutamic acid 183. However, significantly increased enantioselectivity was observed for the epoxidation of bulky styrene derivatives. Furthermore, my study provides strong evidence for the proposed histidine/cysteine ligand switch in chloroperoxidase, providing experimental support for the structure of the 420-nm absorption maximum for a number of carbon monoxide complexes of heme-thiolate proteins. For the NMR study, [dCPO(heme)] was produced using 90% deuterated growth medium with excess heme precursors and [dCPO(Phe)] was grown in the same highly deuterated medium that had been supplemented with excess natural phenylalanine. To make complete heme proton assignments, NMR spectroscopy has been performed for high-resolution structural characterization of [dCPO(heme)] and [dCPO(Phe)] to achieve unambiguous and complete heme proton assignments, which also allows important amino acids close to the heme active center to be determined.

Control of MNC environmental performance and the challenges of subsidiary network dimensions

The trend of green consumerism and increased standardization of environmental regulations has driven multinational corporations (MNCs) to seek standardization of environmental practices or at least seek to be associated with such behavior. In fact, many firms are seeking to free ride on this global green movement, without having the actual ecological footprint to substantiate their environmental claims. While scholars have articulated the benefits from such optimization of uniform global green operations, the challenges for MNCs to control and implement such operations are understudied. For firms to translate environmental commitment to actual performance, the obstacles are substantial, particularly for the MNC. This is attributed to headquarters' (HQ) control challenges (1) in managing core elements of the corporate environmental management (CEM) process and specifically matching verbal commitment and policy with ecological performance and by (2) the fact that the MNC operates in multiple markets and the HQ is required to implement policy across complex subsidiary networks consisting of diverse and distant units. Drawing from the literature on HQ challenges of MNC management and control, this study examines (1) how core components of the CEM process impact optimization of global environmental performance (GEP) and then uses network theory to examine how (2) a subsidiary network's dimensions can present challenges to the implementation of green management policies. It presents a framework for CEM which includes (1) MNCs' Verbal environmental commitment, (2) green policy Management which guides standards for operations, (3) actual environmental Performance reflected in a firm's ecological footprint and (4) corporate environmental Reputation (VMPR). Then it explains how an MNC's key subsidiary network dimensions (density, diversity, and dispersion) create challenges that hinder the relationship between green policy management and actual environmental performance. It combines content analysis, multiple regression, and post-hoc hierarchal cluster analysis to study US manufacturing MNCs. The findings support a positive significant effect of verbal environmental commitment and green policy management on actual global environmental performance and environmental reputation, as well as a direct impact of verbal environmental commitment on green policy management. Unexpectedly, network dimensions were not found to moderate the relationship between green management policy and GEP.

Social-cognitive antecedents of ambidextrous orientation in family-owned startups: The role of family ties, achievement motivation, and internal locus of control

Regulatory Focus Theory predicts that the motivation to self-regulate goal-directed thought and behavior depends on two distinct regulation strategies: a promotion focus based on attaining gains and a prevention focus based on avoiding losses. This study took a social-cognitive approach predicting that regulatory focus has an impact on how family startups (several family related founders) explore "new ideas", exploit "old certainties" and achieve the balance of both (ambidexterity), compared to lone founder startups (only one founder present). It was proposed that the social context of family ties among founders leads them to a prevention focus concerned with avoiding the loss of the socio-emotional benefits of those ties. In order to avoid such a loss, family founders were expected to increase their risk perceptions and thus, explore less than lone founders, who lack such socio-emotional ties. It was also proposed that two commonly used psychological traits in entrepreneurship research —achievement motivation and internal locus of control, predispose entrepreneurs to a promotion focus. Founders with a promotion focus, in turn, were hypothesized to lead startups to more risk-seeking behaviors and to more explorative orientation. The previous argument was used as a springboard to derive hypotheses about ambidexterity (the ability to exploit and explore simultaneously) and survival hazards. Using Regulatory Focus Theory, exploitative orientation, conceptualized as the motivational strength to continue on previous paths of action, was hypothesized to be not significantly different from that of lone founder startups. Taking previous arguments together, lone founder startups were hypothesized to be more ambidextrous than family startups. Finally, ambidexterity and internal locus of control were hypothesized to reduce survival hazards in family startups. The findings suggested that family startups explore less than lone founder startups even after controlling for group effects. Interesting but contradictory findings revealed that internal locus of control have both a positive direct effect and a positive interaction that increases the explorative and ambidextrous orientation gap of family startups over lone founder startups. As expected, ambidexterity and internal locus of control reduced survival hazards on family startups. Implications for practitioners were derived based on a sample of 470 nascent entrepreneurs.

Social-cognitive Antecedents of Ambidextrous Orientation in Family-owned Startups: The Role of Family Ties, Achievement Motivation, and Internal Locus of Control

Regulatory Focus Theory predicts that the motivation to self-regulate goal-directed thought and behavior depends on two distinct regulation strategies: a promotion focus based on attaining gains and a prevention focus based on avoiding losses. This study took a social-cognitive approach predicting that regulatory focus has an impact on how family startups (several family related founders) explore “new ideas”, exploit “old certainties” and achieve the balance of both (ambidexterity), compared to lone founder startups (only one founder present). It was proposed that the social context of family ties among founders leads them to a prevention focus concerned with avoiding the loss of the socio-emotional benefits of those ties. In order to avoid such a loss, family founders were expected to increase their risk perceptions and thus, explore less than lone founders, who lack such socio-emotional ties. It was also proposed that two commonly used psychological traits in entrepreneurship research --achievement motivation and internal locus of control, predispose entrepreneurs to a promotion focus. Founders with a promotion focus, in turn, were hypothesized to lead startups to more risk-seeking behaviors and to more explorative orientation. The previous argument was used as a springboard to derive hypotheses about ambidexterity (the ability to exploit and explore simultaneously) and survival hazards. Using Regulatory Focus Theory, exploitative orientation, conceptualized as the motivational strength to continue on previous paths of action, was hypothesized to be not significantly different from that of lone founder startups. Taking previous arguments together, lone founder startups were hypothesized to be more ambidextrous than family startups. Finally, ambidexterity and internal locus of control were hypothesized to reduce survival hazards in family startups. The findings suggested that family startups explore less than lone founder startups even after controlling for group effects. Interesting but contradictory findings revealed that internal locus of control have both a positive direct effect and a positive interaction that increases the explorative and ambidextrous orientation gap of family startups over lone founder startups. As expected, ambidexterity and internal locus of control reduced survival hazards on family startups. Implications for practitioners were derived based on a sample of 470 nascent entrepreneurs.

Synthesis, characterization and study of physical properties of novel M(n+1)AX(n) compounds

Mn+1AXn compounds, the ternary layered nanolaminates have gathered momentum in the last decade since its advent because of their unusual but exciting properties. These technologically important compounds combine some of the best properties of metals and ceramics. Like ceramics they are refractory, oxidation resistant, elastically stiff and relatively light. They also exhibit metallic properties like excellent machinability, thermal and electrical conductivity. This dissertation concentrates on the synthesis of germanium-based 211 Mn+1AXn compounds. The main objective of the research was to synthesize predominantly single phase samples of Cr2GeC, V2GeC and Ti2GeC. Another goal was to study the effect of solid substitutions on the M-site of Mn+1AXn compounds with Ge as an A-element. This study is in itself the first to demonstrate the synthesis of (Cr0.5V0.5)2GeC a novel Mn+1AXn compound. Scanning electron microscopy coupled with energy dispersive spectroscopy, x-ray diffraction and electron probe microanalysis were employed to confirm the presence of predominantly single phase samples of M2GeC compounds where M = Ti, V, Cr and (Cr 0.5V0.5). A large part of the dissertation also focuses on the effect of the compressibility on the Ge-based 211 Mn+1AXn compounds with the aid of diamond anvil cell and high energy synchrotron radiation. This study also concentrates on the stability of these compounds at high temperature and thereby determines its suitability as high temperature structural materials. In order to better understand the effect of substitutions on A-site of 211 Mn+1 AXn compounds under high pressure and high temperature, a comparison is made with previously reported 211 Mn+1AXn compounds with Al, Ga and S as A-site elements.