The enzyme mechanism of patchoulol synthase

Different mechanisms for the cyclisation of farnesyl pyrophosphate to patchoulol by the patchoulol synthase are discussed in the literature. They are based on isotopic labelling experiments, but the results from these experiments are contradictory. The present work reports on a reinvestigation of patchoulol biosynthesis by isotopic labelling experiments and computational chemistry. The results are in favour of a pathway through the neutral intermediates germacrene A and α-bulnesene that are both reactivated by protonation for further cyclisation steps, while previously discussed intra- and intermolecular hydrogen transfers are not supported. Furthermore, the isolation of the new natural product (2S,3S,7S,10R)-guaia-1,11-dien-10-ol from patchouli oil is reported.

Isotopic Labelling Experiments and Enzymatic Preparation of Iso-Casbenes with Casbene Synthase from Ricinus communis

Isotopic labelling experiments gave insights into the enzyme mechanism of casbene synthase from Ricinus communis, showing a clear stereochemical course for the cyclisation reaction, in agreement with the reported absolute...

A Case Study of the Glycoside Hydrolase Enzyme Mechanism Using an Automated QM-cluster Model Building Toolkit

Glycoside hydrolase enzymes are important for hydrolyzing the β-1,4 glycosidic bond in polysaccharides for deconstruction of carbohydrates. The two-step retaining reaction mechanism was explored with different sized QM-cluster models built by the Residue Interaction Network ResidUe Selector (RINRUS) software using both the wild-type protein and its E217Q mutant. The first step is the glycosylation, in which the acidic residue 217 donates a proton to the glycosidic oxygen leading to bond cleavage. In the subsequent deglycosylation step, one water molecule migrates into the active site and attacks the anomeric carbon. Residue interaction-based QM-cluster models lead to reliable structural and energetic results for proposed glycoside hydrolase mechanisms. The free energies of activation for glycosylation in the largest QM-cluster models were predicted to be 19.5 and 31.4 kcal mol for the wild-type protein and its E217Q mutant, which agree with experimental trends that mutation of the acidic residue Glu217 to Gln will slow down the reaction, and are higher in free energy than the deglycosylation transition states (13.8 and 25.5 kcal mol for the wild-type protein and its mutant, respectively). For the mutated protein, glycosylation led to a low-energy product. This thermodynamic sink may correspond to the intermediate state which was isolated in the X-ray crystal structure. Hence, the glycosylation is validated to be the rate-limiting step in both the wild-type and mutated enzyme. The E217Q mutation led to a higher glycosylation activation free energy that also agrees with experimental observation that mutation of E217 will slow down the reaction, but not deactivate catalysis.

Structure and function of N-acetylglucosamine kinase illuminates the catalytic mechanism of ROK kinases

N-acetyl-D-glucosamine (GlcNAc) is a major component of bacterial cell walls. Many organisms recycle GlcNAc from the cell wall or metabolise environmental GlcNAc. The first step in GlcNAc metabolism is phosphorylation to GlcNAc-6-phosphate. In bacteria, the ROK family kinase NagK performs this activity. Although ROK kinases have been studied extensively, no ternary complex showing the two substrates has yet been observed. Here, we solved the structure of NagK from the human pathogen Plesiomonas shigelloides in complex with GlcNAc and the ATP analogue AMP-PNP. Surprisingly, PsNagK showed two conformational changes associated with the binding of each substrate. Consistent with this, the enzyme showed a sequential random enzyme mechanism. This indicates that the enzyme acts as a coordinated unit responding to each interaction. Molecular dynamics modelling of catalytic ion binding confirmed the location of the essential catalytic metal. Site-directed mutagenesis confirmed the catalytic base, and that the metal coordinating residue is essential. Together, this study provides the most comprehensive insight into the activity of a ROK kinase.

A PCR plus restriction enzyme-based technique for detecting target-enzyme mutations at position Pro-106 in glyphosate-resistant Lolium perenne

The first step in managing herbicide-resistant weeds is to confirm their resistance status. It is, therefore, crucial to have a rapid, reliable and cost-effective technique to assess samples for herbicide resistance. We designed and evaluated three derived cleaved amplified polymorphic sequence (dCAPS) markers for detecting glyphosate resistance in Lolium perenne. conferred by non-synonymous mutations at codon-106 in the enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene. The dCAPS markers involve amplification of the target region, digestion of the amplified products with restriction enzymes and gel-based visualisation of the digested products. The results showed that all three dCAPS markers could successfully detect mutations at codon-106 in the target enzyme. The dCAPS markers can also inform us of the zygosity state of the resistance allele and was confirmed by sequencing the target region of the EPSPS gene. The markers described here are effective quick tests for the monitoring and evaluation of the target-enzyme mechanism of glyphosate resistance in Lolium perenne.

Mechanistic divergence between (4S,7R)-germacra-(1(10)E,5E)-dien-11-ol synthases from Dictyostelium purpureum and Streptomyces coelicolor

The enzyme mechanism of germacradienol synthase DpTPS9 from Dictyostelium purpureum has been studied by labelling experiments and site-directed mutagenesis, and is different to the mechanism of geosmin synthase SCO6073 from Streptomyces coelicolor.

Isoishwarane synthase from Streptomyces lincolnensis

The product of a terpene synthase from Streptomyces lincolnensis has been identified as the new natural product isoishwarane. The enzyme mechanism was studied by isotopic labelling experiments and site-directed mutagenesis.

Solid-state NMR approaches to investigate large enzymes in complex with substrates and inhibitors

Enzyme catalysis is omnipresent in the cell. The mechanisms by which highly evolved protein folds enable rapid and specific chemical transformation of substrates belong to the marvels of structural biology. Targeting of enzymes with inhibitors has immediate application in drug discovery, from chemotherapeutics over antibiotics to antivirals. NMR spectroscopy combines multiple assets for the investigation of enzyme function. The non-invasive technique can probe enzyme structure and dynamics and map interactions with substrates, cofactors and inhibitors at the atomic level. With experiments performed at close to native conditions, catalytic transformations can be monitored in real time, giving access to kinetic parameters. The power of NMR in the solid state, in contrast with solution, lies in the absence of fundamental size limitations, which is crucial for enzymes that are either membrane-embedded or assemble into large soluble complexes exceeding hundreds of kilodaltons in molecular weight. Here we review recent progress in solid-state NMR methodology, which has taken big leaps in the past years due to steady improvements in hardware design, notably magic angle spinning, and connect it to parallel biochemical advances that enable isotope labelling of increasingly complex enzymes. We first discuss general concepts and requirements of the method and then highlight the state-of-the-art in sample preparation, structure determination, dynamics and interaction studies. We focus on examples where solid-state NMR has been instrumental in elucidating enzyme mechanism, alone or in integrative studies.

4. Structure for catalysis

‘Structure for catalysis’ details the various patterns of enzyme mechanism and the various structural features helping to achieve catalysis. One of the striking features of enzyme catalysis is substrate specificity. In the lock-and-key hypothesis, the enzyme is viewed as a precisely shaped lock and only the right key, the substrate, can fit and turn it. The lock-and-key combination is the enzyme–substrate complex. A crucial ingredient of the enzyme’s equipment for achieving outstanding catalysis is the ‘catalytic groups’.

Room-temperature X-ray crystallography reveals the oxidation and reactivity of cysteine residues in SARS-CoV-2 3CL Mpro: insights into enzyme mechanism and drug design

The emergence of the novel coronavirus SARS-CoV-2 has resulted in a worldwide pandemic not seen in generations. Creating treatments and vaccines to battle COVID-19, the disease caused by the virus, is of paramount importance in order to stop its spread and save lives. The viral main protease, 3CL Mpro, is indispensable for the replication of SARS-CoV-2 and is therefore an important target for the design of specific protease inhibitors. Detailed knowledge of the structure and function of 3CL Mpro is crucial to guide structure-aided and computational drug-design efforts. Here, the oxidation and reactivity of the cysteine residues of the protease are reported using room-temperature X-ray crystallography, revealing that the catalytic Cys145 can be trapped in the peroxysulfenic acid oxidation state at physiological pH, while the other surface cysteines remain reduced. Only Cys145 and Cys156 react with the alkylating agent N-ethylmaleimide. It is suggested that the zwitterionic Cys145–His45 catalytic dyad is the reactive species that initiates catalysis, rather than Cys145-to-His41 proton transfer via the general acid–base mechanism upon substrate binding. The structures also provide insight into the design of improved 3CL Mpro inhibitors.