The dynamics of complex cellular machinery required for methionine synthesis in mammalian cells

Humans, like all living things, have specialised molecules for carrying out specific biological functions. Some of these functions are executed by specifically designed enzymes. One enzyme in particular, called methionine synthase, is quite extraordinary on a number of levels. First, it utilizes vitamin B12 to function; it is only one of two human enzymes to exploit its highly reactive properties. Oxygen, however, can damage vitamin B12, and prevent methionine synthase from carring out its biological function. Fortunately, humans have another enzyme, called methionine synthase reductase that repairs vitamin B12 and restores methionine synthase activity. Second, methionine synthase has an important role in human health, as it reduces the amount of a toxic compound in the body that can damage cells and cause cardiovascular disease. The enzyme also produces compounds that are essential for making DNA and protein. Impairment of the enzyme's activity prevents the ability of cells to divide and grow. Third, methionine synthase is very complex structurally, as it is composed of four individual rigid units that are linked together by flexible connectors. Two of the units bind to substrate molecules, another houses vitamin B12 and the fourth interacts with the repair enzyme, methionine synthase reductase. For the enzyme to function, all four units must move in specific order at a specific time over relatively large distances. Moreover, methionine synthase reductase is also composed of mobile units that must engage and disengage with each other and with specific units of methionine synthase at key times. The complexity associated with coordinating movement of these units is rare amongst enzymes. We want to measure the distance between individual units at specific times, and determine how far and how fast they travel. We also want to understand what controls movement of these individual units and how their movement is synchronised. To address these questions, we will determine the molecular architecture of both enzymes, (i.e. where each atom of the enzyme is located in a three dimensional space), as individual entities and when they are bound to each other. We will also try to solve the molecular architecture of the individual units. From this information, we will be able to examine unique structural features of the enzymes that are associated with movement. To measure distance between units at key times, we will use different techniques for each enzyme. Methionine synthase reductase contains intrinsic probes one in each of the mobile units that can be used to measure distance between the units. Under a certain physical state, these two probes contain an electron that acts like a miniature magnet. Surrounded by a larger magnet and a microwave frequency, these probes can report on their immediate environment, (i.e. neighbouring atoms) as well as the distance to each other. Methionine synthase does not contain intrinsic probes; therefore, we will attach them artificially to the surface of the enzyme. By shining laser light at a particular frequency, energy is transferred from one probe to the next. However, the amount of energy transferred is dependent on the distance between the probes. Since we are able to measure the energy transferred over time, we can observe the time-dependent distance between the probes. Moreover, we will have the capability to observe single enzyme molecules, and we will be able to determine if they behave similarly or not. This relatively new technique can reveal a wealth of information of the structural and functional properties of enzymes. This will be the first time it is applied to such a complex enzyme system.

It is becoming increasingly apparent that linkers (peptide sequences that tether larger function/catalytic domains) have major roles in choreographing movement of large modular proteins. To date, the role of these short interspacing sequences in macromolecular assemblies has focused on gene regulation and signal transduction; metabolic processes are generally overlooked, the exceptions being polyketide synthases. The MS/MSR system is a large multi-protein catalytic complex, central to metabolism and homoeostasis, in which domain motion is coupled to the catalytic chemistry. It is an ideal model for investigating the dynamics of macromolecular assemblies in the metabolic realm. Both MS and MSR are large modular proteins with large inter domain linkers. The individual modules within both enzymes must act in a highly orchestrated manner to carry out three separate transmethylation reactions in the case for MS, or electron transfer in the case of MSR. Additionally, these two modular proteins must assemble with each other during the cob(II)alamin regeneration process. For MS in particular, domain movement must be carefully synchronized as the enzyme houses, at least during part of the catalytic cycle, a reactive cob(I)alamin. Exposure of this powerful nucleophile to the cell milieu leads to MS inactivation with consequent effects on cell homeostasis. To understand the crucial role of linkers in establishing structural and functional assembly of the multi-modular MS/MSR system, we need information on the structure and mechanism of individual components, the full length proteins and a detailed 'dynamic profile' for the assembled complex. We thus propose an interdisciplinary approach involving a combination of x-ray crystallography, electron magnetic resonance spectroscopy and single pair fluorescence energy transfer (spFRET) measurements to investigate how large-scale inter and intra molecular domain movement is synchronized in catalysis and during enzyme reactivation.