Enzyme promiscuity and the origins of cellular innovations : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Albany, New Zealand

Biochemistry textbooks define enzymes as being efficient and highly specific. However, these characteristics are usually associated with a lack of versatility, and therefore, an inability to evolve new functions. In spite of this, it is known that new enzymes can arise rapidly (such as when bacteria evolve antibiotic resistance). One hypothesis proposes that enzymes are actually promiscuous (Jensen, 1976); that is, they are able to carry out secondary reactions, in addition to the one they evolved to catalyze. The goal of this research was to explore the role that promiscuity plays in the origins and evolution of enzyme functions, using Escherichia coli as a model organism. In the first part of this thesis, I report the discovery of two enzymes (alanine racemase and cystathionine ß-lyase) that are reciprocally promiscuous, and are dependent on the cofactor pyridoxal 5’-phosphate (PLP) for activity. In vivo, the cofactor-mediated promiscuous activities of alanine racemase and cystathionine ß-lyase were each successfully improved to near wildtype levels using directed evolution experiments. These results extend Jensen’s hypothesis, and led me to propose that PLP played a significant role in the evolution of new enzymes, in the primordial world. In the second part of the thesis, I developed a comprehensive library-on-library screen to search for E. coli proteins that could mediate improved growth in environments containing either a foreign nutrient or a toxin. Proteins were over-expressed in an attempt to increase their weak, promiscuous activities, and to mimic the common genetic phenomenon of gene amplification. Over-expression of individual proteins conferred improved growth to the host cell in 35% of ~2,000 environments. The findings have important implications for understanding bacterial adaptation to new environments, such as when antibiotic resistance emerges. The ability of promiscuous proteins to drive the emergence of new phenotypes, when their expression is increased, validates the feasibility of the Innovation, Amplification and Divergence (IAD) model for the evolution of new genes (Bergthorsson et al., 2007). Overall, the work described in this thesis demonstrates that protein promiscuity is common, though difficult to predict a priori. My experimental results are consistent with the work of others, in suggesting that promiscuous activities are evolvable. Together, the high frequency and evolvability of promiscuous proteins appear to underpin many different cellular innovations

Are Malaysian exports and imports cointegrated?

This paper investigates the long-run relationship between Malaysian exports and imports. Towards this end, multivariate cointegration techniques have been applied. For all measures (or cases), both the unrestricted and restricted cointegration results demonstrate that there exists a long-run relationship between Malaysian exports and imports. Simply put, both the variables will converge towards equilibrium in the long run which indicates the effectiveness of Malaysia?s long-term macroeconomic planning in stabilising trade balance. Thus, the Malaysian economy does not violate its inter-temporal budget constraint

Production of Ethylene From Ethanol Dehydration Over H3PO4-Modified Cerium Oxide Catalysts = Penghasilan Etilena Daripada Pendehidratan Etanol Dengan Mangkin Serium Oksida Terubahsuai H3PO4

Production of ethylene from ethanol dehydration was investigated over H3PO4 (10 wt.% to 30wt.%)-modified cerium oxide catalysts synthesized by wet impregnation technique. The prepared catalysts were characterized using scanning electron microscope (SEM), N2 adsorption-desorption method, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA) for the physicochemical properties. The ethanol catalytic dehydration was carried out in a fixed-bed reactor at 673-773 K and at ethanol partial pressure of 33 kPa. The effects of phosphorus loading on catalyst and reaction temperatures were investigated in terms of catalytic activity towards product selectivity and yield. Overall, the selectivity and yield of ethylene increased with the temperature and phosphorus loading. The highest ethylene selectivity and yield were 99% and 65%, respectively, at 773 K and 33 kPa over the 30 wt.% H3PO4-modified cerium oxide

Self-Dual Conformal Supergravity and the Hamiltonian Formulation

In terms of Dirac matrices the self-dual and anti-self-dual decomposition of a conformal supergravity is given and a self-dual conformal supergravity theory is developed as a connection dynamic theory in which the basic dynamic variabes include the self-dual spin connection i.e. the Ashtekar connection rather than the triad. The Hamiltonian formulation and the constraints are obtained by using the Dirac-Bergmann algorithm. PACS numbers: 04.20.Cv, 04.20.Fy,04.65.+

Molecularly targeted therapies for asthma: current development, challenges and potential clinical translation

Extensive research into the therapeutics of asthma has yielded numerous effective interventions over the past few decades. However, adverse effects and ineffectiveness of most of these medications especially in the management of steroid resistant severe asthma necessitate the development of better medications. Numerous drug targets with inherent airway smooth muscle tone modulatory role have been identified for asthma therapy. This article reviews the latest understanding of underlying molecular aetiology of asthma towards design and development of better antiasthma drugs. New drug candidates with their putative targets that have shown promising results in the preclinical and/or clinical trials are summarised. Examples of these interventions include restoration of Th1/Th2 balance by the use of newly developed immunomodulators such as toll-like receptor-9 activators (CYT003-QbG10 and QAX-935). Clinical trials revealed the safety and effectiveness of chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) antagonists such as OC0000459, BI-671800 and ARRY-502 in the restoration of Th1/Th2 balance. Regulation of cytokine activity by the use of newly developed biologics such as benralizumab, reslizumab, mepolizumab, lebrikizumab, tralokinumab, dupilumab and brodalumab are at the stage of clinical development. Transcription factors are potential targets for asthma therapy, for example SB010, a GATA-3 DNAzyme is at its early stage of clinical trial. Other candidates such as inhibitors of Rho kinases (Fasudil and Y-27632), phosphodiesterase inhibitors (GSK256066, CHF 6001, roflumilast, RPL 554) and proteinase of activated receptor-2 (ENMD-1068) are also discussed. Preclinical results of blockade of calcium sensing receptor by the use of calcilytics such as calcitriol abrogates cardinal signs of asthma. Nevertheless, successful translation of promising preclinical data into clinically viable interventions remains a major challenge to the development of novel anti-asthmatics

Catalytic Performance of Commercial Zeolites Y as Catalyst for Ethylene Production from Ethanol Dehydration

Catalytic dehydration of ethanol into ethylene was studied over commercial Zeolites-Y with different Si:Al ratios between 5.1:1 and 80:1, and temperature from 573 K to 773 K. The physicochemical properties of fresh and spent catalyst of Zeolite Y Si:Al 80:1 (best performing catalyst) were investigated using N2-physisorption, TGA, SEM-EDX, NH3-TPD, FTIR and XRD. Results showed that catalysts with higher Si:Al ratios exhibit better catalytic performance in terms of higher ethanol conversion and higher selectivity to ethylene. Indeed, zeolites-Y with Si:Al ratio 5.1:1 and 12:1 demonstrated low catalytic activity with ethanol conversion of 34% and 2%, respectively. However, ethylene selectivity of NH3-Y (5) was 84%, which was considerably higher than NH3-Y (12) which was 26%, indicated that this catalyst was not promoting the formation of other hydrocarbons i.e. methane and ethane. Albeit all of the catalysts namely H-Y (30), H-Y (60) and H-Y (80) showed favorable performance in ethanol dehydration, H-Y (80) attained almost total selectivity to ethylene and highest conversion of 73.0% among all the tested catalysts

IL-8 as a potential in-vitro severity biomarker for dengue disease

Dengue is a common infection, caused by dengue virus. There are four different dengue serotypes, with different capacity to cause severe dengue infections. Besides, secondary infections with heterologous serotypes, concurrent infections of multiple dengue serotypes may alter the severity of dengue infection. This study aims to compare the severity of single infection and concurrent infections of different combinations of dengue serotypes in-vitro. Human mast cells (HMC)-1.1 were infected with single and concurrent infections of multiple dengue serotypes. The infected HMC-1.1 supernatant was then added to human umbilical cord vascular endothelial cells (HUVEC) and severity of dengue infections was measured by the percentage of transendothelial electrical resistance (TEER). Levels of IL- 10, CXCL10 and sTRAIL in HMC-1.1 and IL-8, IL-10 and CXCL10 in HUVEC culture supernatants were measured by the ELISA assays. The result showed that the percentage of TEER values were significantly lower in single infections (p0.4), indicating that IL-8 may be suitable as an in-vitro severity biomarker. In conclusion, this in-vitro model presented few similarities with regards to the conditions in dengue patients, suggesting that it could serve as a severity model to test for severity and levels of severity biomarkers upon different dengue virus infections