School of Chemistry - Theses
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ItemDevelopment of a molecular description of the Embden-Meyerhof-Parnas sulfoglycolysis pathway( 2019)Sulfoquinovose (SQ, 6-deoxy-6-sulfoglucose) is the polar head group of the plant sulfolipid, sulfoquinovoside diacylglycerol (SQDG). SQ is estimated to be synthesized at a scale of some 10 billion tonnes annually and is a major contributor to the biogeochemical sulfur cycle. SQDG is a thylakoid membrane sulfolipid and is involved in membrane structure and function, and modulates the activity of photosynthetic complexes. Two sulfoglycolysis pathways have been discovered in recent years that allow the degradation of SQDG: the sulfoglycolytic Embden-Meyerhof-Parnas (sulfo-EMP) pathway and the sulfoglycolytic Entner-Doudoroff (sulfo-ED) pathway. SQDG hydrolysis to form SQ, enabling entry of SQDG to the sulfur cycle, is catalyzed by glycosidases termed sulfoquinovosidases (SQases). Structural analysis of the first SQase, EcYihQ from Escherichia coli, revealed substrate recognition by a QQRWY motif in the active site. Other putative sulfoquinovosidases contain a KERWY motif; it is not known whether they are genuine sulfoquinovosidases. In Chapter 2 we present the discovery and characterization of a second sulfoquinovosidase that bears a KERWY motif, AtSQase from Agrobacterium tumefaciens. AtSQase catalyzes hydrolysis of the artificial substrate p-nitrophenyl sulfoquinovoside (PNPSQ), which enabled its kinetic and structural characterization. Through the synthesis of a series of analogues of PNPSQ it is shown that EcYihQ and AtSQase are highly specific for both correct substrate stereochemistry and the sulfonate group. The first SQase inhibitor SQ-IFG was designed and synthesized. Structural analysis of both enzymes allowed the identification of catalytic and substrate binding residues. Their roles were supported by mutagenesis and kinetic analysis. Mutual information analysis provided insights into the evolution of these proteins. Chapter 3 covers studies of sulfoquinovose mutarotase activity using a homolog of the putative mutarotase YihR from E. coli, namely the sulfoquinovose mutarotase from Herbaspirillum seropedicae (HsSQM). With the use of 1D and 2D EXSY NMR techniques, unidirectional mutarotation rates in equilibrium mixtures of various hexoses, including SQ were measured. The enzyme exhibited a broad spectrum mutarotase activity but did not tolerate an axial C2 hydroxyl group. Further studies demonstrated that this enzyme is a sulfoquinovose mutarotase with approximately 17 000-fold preference for SQ compared to glucose 6-phosphate. Chapter 4 explored the catalytic activity of E. coli YihS catalyzing isomerization of SQ and 6-deoxy-6-sulfofructose (SF). Both substrates for the enzyme were synthesized, and NMR studies demonstrated the reversible interconversion of SQ to SF with formation of a new product, sulforhamnose (SR, 6-deoxy-6-sulfomannose). HPLC analysis showed that EcYihS catalysed the isomerisation of SQ at a rate approximately 178-fold greater compared to D-mannose (a previously described substrate for this enzyme), in terms of kcat/KM. NMR studies of the rate of YihS catalyzed H/D isotope exchange revealed that EcYihS prefers beta-SQ as the substrate. Chapter 5 of the thesis focused on the EcYihU catalyzed reduction of sulfolactaldehyde (SLA) to dihydroxypropane-1-sulfonate (DHPS). The substrate SLA was synthesized and its stability was defined. By monitoring consumption of cofactor, the rate of EcYihU catalyzed conversion of SLA to DHPS was measured, showing higher activity compared to succinic semialdehyde, a previously described substrate for this enzyme. Reduced forms of the cofactor NADH were synthesized; analysis of their inhibitory potency revealed that tetrahydro-NADH was more potent than hexahydro-NADH. Mechanistic studies using these inhibitors supported a sequential kinetic mechanism for EcYihU. X-ray studies have identified the sulfonate binding residues and revealed domain movements in YihU upon substrate/cofactor binding.