<p>HMG-CoA reductase from Pseudomonas mevalonii (<i>Pm</i>HMGR) catalyzes the oxidation of mevalonate and mevaldyl-CoA to form HMG-CoA using CoA-SH and two NAD+ cofactors. While the enzyme has been used extensively as a drug target in humans to treat hypercholesterolemia, its pathway has also been found to be critical for the survival of antibiotic resistant gram-positive bacteria. Structural studies using non-productive and slow-substrate binary complexes as well as biochemical studies using half and full reactions led to the proposal that the conversion of mevalonate to HMG-CoA occurs through the generation of two intermediates, mevaldehyde and mevaldyl-CoA (Shown in Fig 1.1). However, several intermediary changes along the <i>Pm</i>HMGR reaction pathway remain unclear. By gathering information about the enzyme’s intermediate states via structural studies, we could identify potential allosteric sites that further the reaction mechanism. Using this knowledge, we could design enzyme inhibitors that act as novel antibacterials. The application of time-resolved crystallographic methods would provide structural information about transitory states in the PmHMGR reaction mechanism. The <i>Pm</i>HMGR crystal has been shown to be suitable for time-resolved crystallographic measurements for the reaction steps resulting in mevaldyl-CoA formation. However, our structural investigations of the mevalonate, CoA and NAD+ complex that are expected to result in the formation of mevaldehyde (Fig 1.1) do not show any changes corresponding to a turnover in the crystal environment. <br></p><p><br></p><p>To investigate the factors limiting enzymatic activity in the crystal, we investigated the effects of pH and specific ions in the crystallization environment. Kinetic studies indicated a strong <i>Pm</i>HMHGR inhibition in the crystallization buffer that is dependent on the concentration of the crystallization precipitant ammonium sulfate. These studies also indicated an increase in enzyme turnover with increasing pH. Utilizing the ionic concentration and pH-dependent properties of the enzyme in the crystallization environment, we have developed a reaction triggering approach using pH changes for <i>Pm</i>HMGR crystals.<br></p><p><br></p><p>We have demonstrated our application of this ‘pH-jump’ method by observing changes in <i>Pm</i>HMGR crystals after reaction initiation. Changes in the density of mevalonate, CoA and NAD+have indicated mevaldehyde and mevaldyl-CoA formation. Additionally, the appearance of a unique NADH absorbance peak after the pH-change has also highlighted the initiation of the <i>Pm</i>HMGR reaction and the occurrence of a hydride transfer step. Our analysis of the movements using time-resolved structures post reaction-initiation have also highlighted structural changes and inter-domain contacts in the small and flap domain that would allow cofactor exchange and product release. The pH-jump method can hence be utilized as a novel approach for triggering the <i>Pm</i>HMGR reaction in crystals and further studying transitory states along its reaction pathway.<br></p>

Starch is a major carbon and energy source throughout all kingdoms of life. It consists of two carbohydrate polymers, branched amylopectin and linear amylose, which are sparingly soluble in water. Hence, the enzymatic breakdown by glycoside hydrolases (GHs) is of great biological and societal importance. Amylomaltases (AMs) are GHs specialized in the hydrolysis of a-1,4–linked sugar chains such as amylose. They are able to catalyze an intramolecular transglycosylation of a bound sugar chain yielding polymeric sugar rings, the cycloamyloses (CAs), consisting of 20 to 100 glucose units. Despite a wealth of data on short oligosaccharide binding to GHs, no structural evidence is available for their interaction with polymeric substrates that better represent the natural polysaccharide. We have determined the crystal structure of Thermus aquaticus AM in complex with a 34-meric CA—one of the largest carbohydrates resolved by x-ray crystallography and a mimic of the natural polymeric amylose substrate. In total, 15 glucose residues interact with the protein in an extended crevice with a length of more than 40 Å. A modified succinimide, derived from aspartate, mediates protein-sugar interactions, suggesting a biological role for this nonstandard amino acid. The structure, together with functional assays, provides unique insights into the interaction of GHs with their polymeric substrate and reveals a molecular ruler mechanism for minimal ring-size determination of CA products.