I formally joined Prof. M. R. N. Murthy’s laboratory at the Molecular Biophysics
Unit, Indian institute of Science, on 1st August 2001. During that time, the interest in the laboratory was mainly focused on structural studies on a number of capsid mutants of two plant viruses, sesbania mosaic virus and physalis mottle virus, to gain an insight into the virus structure and its assembly. Besides these two projects, there were a few other collaborative projects running in the lab at that time such as NIa protease from pepper vein banding virus and diaminopropionate ammonia lyase from Escherichia coli with Prof. H. S. Savithri, triosephosphate isomerase from Plasmodium falciparum with Prof. P. Balaram and Prof. H. Balaram and a DNA binding protein (TP2) with Prof. M. R. S. Rao. During my first semester, along with my course work, I was assigned to make an
attempt to purify and crystallize recombinant NIa protease and TP2 protein. I started with NIa protease which could be purified using one step Ni-NTA affinity column chromatography. Although the expression and protein yield were reasonably good, protein precipitated with in a couple of hours after purification. Attempts were made to prevent the precipitation of the purified enzyme and towards this end we were successful to some extent. However, during crystallization trials most of the crystallization drops precipitated completely even at low protein oncentration. TP2 protein was purified using three-step chromatographic techniques by one of the project assistant in Prof. M. R. S. Rao’s laboratory. Because of low expression level and three step purification protocol, protein yield was not good enough for complete crystallization screening. Hits obtained from our initial screening could not be confirmed because of low protein yield as well as batch to batch variation. My attempts to crystallize these two proteins remained unsuccessful but in due course I had learnt a great deal about the tips and tricks of expression, purification and mainly crystallization. To overcome the problems faced with these two proteins, we decided to make some changes in the gene construct and try different expression systems.
By this time (beginning of 2002), I had finished my first semester and a major
part of the course work, so we decided to start a new project focusing on some of the
unknown enzymes from a metabolic pathway. Dr. Parthasarathy, who had finished his
Ph. D. from the lab, helped me in literature work and in finding targets for structural
studies. Finally, we decided to target enzymes involved in the propionate etabolism.
The pathways for propionate metabolism in Escherichia coli as well as Salmonella
typhimurium were just established and there were no structural information available for
most of the enzymes involved in these pathways. Since, propionate metabolic pathways were well described in the case of Salmonella typhimurium, we decided to use this as the model organism. We first started with the enzymes present in the propionate catabolic pathway “2-methylcitrate pathway”, which converts propionate into pyruvate and
succinate. 2-methylcitrate pathway resembles the well-studied glyoxylate and TCA cycle.
Most of the enzymes involved in 2-methylcitrate pathway were not characterized
biochemically as well as structurally. First, we cloned all the four enzymes PrpB, PrpC, PrpD and PrpE present in the prpBCDE operon along with PrpR, a transcription factor, with the help of Dr. P.S. Satheshkumar from Prof. H. S. Savithri’s laboratory. Since these five proteins were cloned with either N- or C-terminal hexa-histidine tag, they could be purified easily using one-step Ni-NTA affinity column chromatography. PrpB, PrpC and PrpD had good expression levels but with PrpE and PrpR, more than 50% of the expressed protein went into insoluble fraction, probably due to the presence of membrane spanning domains in these two enzymes. Around this time, crystallization report for the PrpD from Salmonella was published by Ivan Rayment’s group, so after that we focused only on the remaining four proteins leaving out PrpD. Our initial attempts to crystallize
these proteins became successful in case of PrpB, 2-methylisocitrate lyase. We collected
a complete diffraction data to a resolution of 2.5 Å which was later on extended to a
resolution of 2.1 Å using another crystal. Repeated crystallization trials with PrpC also gave small protein crystals but they were not easy to reproduce and size and diffraction quality always remained a problem. Using one good crystal obtained for PrpC, data to a resolution of 3.5 Å could be collected. Unfortunately, during data collection due to failure of the cryo-system, a complete dataset could not be collected. Further attempts to crystallize this protein made by Nandashree, one of my colleagues in the lab at that time, was also without much success. Attempts to purify and crystallize PrpE and PrpR were made by me as well as one of my colleagues, Anupama. In this case, besides crystallization, low expression and precipitation of the protein after purification were major problems.
Our attempt to phase the PrpB data using the closest search model (phosphoenolpyruvate mutase) by molecular replacement technique was unsuccessful,probably because of low sequence identity between them (24%). Further attempts were made to obtain heavy atom derivatives of PrpB crystal. We could obtain a mercury derivative using PCMBS. However, an electron density map based on this single derivative was not nterpretable. Around this time, the structure of 2-methylisocitrate lyase (PrpB) from E. coli was published by Grimm et. al. The structure of Salmonella PrpB could easily be determined using the E. coli PrpB enzyme as the starting model. We also solved the structure of PrpB in complex with pyruvate and Mg2+. Our attempts to crystallize PrpB with other ligands were not successful. Using the structures of PrpB and its complex with pyruvate and Mg2+, we carried out comparative studies with the well-studied structural and functional homologue, isocitrate lyase. These studies provided the
plausible rationale for different substrate specificities of these two enzymes. Due to
unavailability of PrpB substrate commercially and the extensive biochemical and mutational studies carried out by two different groups made us turn our attention to other enzymes in this metabolic pathway. Since our repeated attempts to obtain good
diffraction quality crystals of PrpC, PrpE and PrpR continued to be unsuccessful, we
decided to target other enzymes involved in propionate metabolism.
We looked into the literature for the metabolic pathways by which propionate is
synthesized in the Salmonella typhimurium and finally decided to target enzymes present
in the metabolic pathway which converts L-threonine to propionate. Formation of
propionate from L-threonine is the most direct route in many organisms. During February 2003, we initiated these studies with the last enzyme of this pathway, propionate kinase (TdcD), and within a couple of months we could obtain a well-diffracting crystal in complex with ADP and with a non-hydrolysable ATP analog, AMPPNP. TdcD structure was solved by molecular replacement using acetate kinase as a search model. Propionate kinase, like acetate kinase, contains a fold with the topology βββαβαβα, identical with that of glycerol kinase, hexokinase, heat shock cognate 70 (Hsc70) and actin, the superfamily of phosphotransferases. Examination of the active site pocket in propionate kinase revealed a plausible structural rationale for the greater specificity of the enzyme
towards propionate than acetate.
One of the datasets of TdcD obtained in the presence of ATP showed extra continuous density beyond the γ-phosphate. Careful examination of this extra electron
density finally allowed us to build diadenosine tetraphosphate (Ap4A) into the active site pocket, which fitted the density very well. Since the data was collected at a synchrotron source to a resolution of 1.98 Å, we could identify the ligand in the active site pocket solely on the basis of difference Fourier map. Later on, co-crystallization trials of TdcD with commercially available Ap4A confirmed its binding to the enzyme. These studies
suggested the presence of a novel Ap4A synthetic activity in TdcD, which is further being examined by biochemical experiments using mass-spectrometry as well as thin-layer chromatography experiments.
By the end of 2004, we shifted our focus to the first enzyme involved in the anaerobic degradation of L-threonine to propionate, a biodegradative threonine deaminase (TdcB). Sagar Chittori, who had joined the lab as an integrated Ph. D student, helped me in cloning this enzyme. My attempt to crystallize this protein became finally
successful and datasets in three different crystal forms were collected. Dataset for TdcB in complex with CMP was collected during a synchrotron trip to SPring8, Japan by my colleague P. Gayathri and Prof. Murthy. TdcB structure was solved by molecular replacement using the N-terminal domain of biosynthetic threonine deaminase as a search model. Structure of TdcB in the native form and in complex with CMP helped us to understand several unanswered questions related to ligand mediated oligomerization and enzyme activation observed in this enzyme.
The structural studies carried out on these three enzymes have provided structural
as well as functional insights into the catalytic process and revealed many unique features of these metabolic enzymes. All these have been possible mainly due to proper guidance and encouragement from Prof. Murthy and Prof. Savithri. Prof. Murthy’s teaching as well as discussions during the course of investigation has helped me in a great deal to learn and understand crystallography. Collaboration with Prof. Savithri kept me close to biochemistry and molecular biology, the background with which I entered the world of structural biology. The freedom to choose the project and carry forward some of my own ideas has given me enough confidence to enjoy doing research in future.
| Identifer | oai:union.ndltd.org:IISc/oai:etd.ncsi.iisc.ernet.in:2005/329 |
| Date | 09 1900 |
| Creators | Simanshu, Dhirendra Kumar |
| Contributors | Murthy, M R N |
| Source Sets | India Institute of Science |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
| Rights | I grant Indian Institute of Science the right to archive and to make available my thesis or dissertation in whole or in part in all forms of media, now hereafter known. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. |
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