Coenzyme recognition in para-hydroxybenzoate hydroxylase

Biochemistry is the science that studies the chemistry of life. This 'biological' chemistry includes growth, differentiation, movement, conductivity, immunity, transport and storage. During these processes proteins play an important role. The building blocks of proteins are amino acids, of which twenty are known. With these building blocks at hand it is possible to construct numerous proteins with many specific functions. A protein is not an elongated chain of amino acid residues but a compact very well defined three-dimensional structure. Two basic substructures are known in a protein, a cylindricalα-helix and an elongatedβ-strand. A number of theseα-helices and/orβ-strands connected by loop regions form a protein domain and a protein is built up of one or more domains. Furthermore, proteins can contain certain motifs (folds), structural conserved patterns. A large group of proteins with similar function and/or structure are called a protein family.A special group of proteins, called enzymes or biocatalysts, are able to increase the rate of a chemical reaction by lowering the activation energy of that reaction. Enzymes are highly specific, because they influence the reactivity of the substrate in such a way that the substrate is quickly and efficiently converted into a product. Moreover, flexible/dynamic movements in enzymes may play an important role during catalysis, because enzymes are not always rigid bodies. To control the reaction, enzymes often need cofactors. Some examples are the already mentioned dinucleotides NAD(P)H and FAD, that play a role in electron transfer (redox) reactions. Generally speaking, these cofactors bind very specific to a protein. A well-known binding motif for NAD(P)H and FAD in different enzyme families is the Rossmann fold (Chapter 1), discovered by Michael Rossmann in 1974.The NAD(P)H cofactor binds to the enzyme, electron transfer takes place and finally, the oxidized cofactor is released. In some proteins, the mode of NADPH binding is unknown. One example is p -hydroxybenzoate hydroxylase (PHBH), a flavoprotein monooxygenase that belongs to the family of FAD-dependent aromatic hydroxylases.FAD-dependent Aromatic HydroxylasesFAD-dependent aromatic hydroxylases play a role in the biodegradation of aromatic compounds. In nature, these compounds occur in plant polymers (lignin) as well as in proteins, steroïds and terpenes. During this century, the natural pool of aromatic compounds has been extended with products of industrial origin. Many of these synthetic compounds (pesticides, herbicides, fungicides and detergents) place a heavy burden on the environment and accumulate in soil and sludge. Microbial FAD-dependent aromatic hydroxylases catalyze the conversion of natural and synthetic aromatic substrates into products that can be further degraded to carbon dioxide and water. Recently, it was found that these enzymes are also involved in the biosynthesis of steroïds, plant hormones and antibiotics. PHBH is the archetype (prototype) of the family of FAD-dependent aromatic hydroxylases. In Wageningen, research on PHBH and related enzymes is embedded in the Wageningen Graduate School of Environmental Chemistry & Toxicology.p -Hydroxybenzoate Hydroxylasep -Hydroxybenzoate hydroxylase is isolated from the soil bacterium Pseudomonas fluorescens . This microbe can grow on 4-hydroxybenzoate (POHB) and other aromatic compounds as sole carbon source. PHBH catalyzes the conversion of POHB into 3,4-dihydroxybenzoate (DOHB) in the presence of NADPH and molecular oxygen. DOHB is a common intermediate in the aerobic degradation of plant material. After ring cleavage of DOHB and further degradation, the final products acetyl coenzyme A and succinate are fed into the citric acid cycle to provide energy for the cell.p -Hydroxybenzoate hydroxylase has been subject to detailed kinetic and structural studies. The three-dimensional structure of PHBH is built up of three domains (Chapter 1). The first domain is the FAD-binding domain with the specific Rossmann fold for binding the ADP part of FAD. The second domain is the substrate-binding domain and the third domain (interface domain) is important for the interaction with another PHBH subunit, because PHBH exists as a dimer.The structure of the enzyme-substrate complex is known in atomic detail. Recently, it was found that the flavin ring is able to move between an "open" and "closed" conformation. This flavin mobility is important for substrate binding and product release. However, unknown is the NADPH-binding site and where the reaction between NADPH and FAD takes place. Related questions are:-Which amino acids play a role in cofactor binding?-Is there a particular sequence motif for cofactor binding?Which amino acids are responsible for the coenzyme specificity and involved in binding of the 2'-phosphate moiety of NADPH?Another very important question concerns the effector role of the substrate. Upon binding of the aromatic substrate the flow of electrons from NADPH to FAD is 105 times enhanced. However, the molecular principles of this control are poorly understood. In this thesis we have tried to shed more light on the coenzyme recognition by PHBH.Flavin ring mobilityIn Chapter 2 the FAD in PHBH is substituted by a modified FAD, normally present in alcohol oxidase from methylotrophic yeasts. The crystal structure of p -hydroxybenzoate hydroxylase with this flavin analog not only represents the first crystal structure of an enzyme reconstituted with a modified flavin, but also provides direct evidence for the presence of an arabityl sugar chain in the modified form of FAD. The reconstituted enzyme-substrate complex shows that the flavin ring attains the "open" conformation. In the native enzyme-substrate complex the flavin ring is located in the "closed" conformation. The rate of flavin reduction by NADPH is much more rapid as compared to the native enzyme-substrate complex, suggesting that the mobility of the flavin ring is essential for the efficient reduction of the enzyme/substrate complex.Amino acids involved in NADPH bindingTo investigate the mode of NADPH binding, several amino acid residues were replaced by site-directed mutagenesis. The amino acids were selected on the basis of earlier results from chemical modification, crystallographic and modeling studies. Chapters 3, 4, 6 and 8 describe the properties of single mutants. It is concluded that Arg33, Gln34, Tyr38, Arg42, Arg44, His162 and Arg269 are involved in NADPH binding.Structural motif for NADPH bindingPHBH contains two conserved sequence motifs, both involved in FAD binding. Chapter 5 describes a new unique sequence motif for the family of FAD-dependent aromatic hydroxylases, putatively involved in both FAD and NAD(P)H binding. From the recently determined crystal structure of phenol hydroxylase it is deduced that this sequence motif is also structurally conserved. Chapter 6 and 7 show that only His162 of this novel motif is directly important for the binding of NADPH.Coenzyme specificityChapter 8 describes the cloning, purification and characterization of PHBH from Pseudomonas species CBS3. This is the first PHBH enzyme with known sequence that is active with NADH. Based on sequence analysis and homology modelling it is proposed that the helix H2 region is important for the binding of the 2'-phosphate moiety of NADPH. In Chapter 9 , the coenzyme specificity of PHBH from Pseudomonas fluorescens was addressed in further detail. Multiple replacements in helix H2 showed that Arg33 and Tyr38 are crucially involved in determining the coenzyme specificity. For the first time, a PHBH enzyme was constructed, which is more efficient with NADH.Effector specificitySubstrate binding is essential for a rapid reduction of FAD. This allows the subsequent attack of oxygen and the formation of the flavinhydroperoxide hydroxylating species. The question arises whether the stimulating effect of substrate binding on flavin reduction is caused by a large conformational change or merely due to subtle rearrangements in the active site. Chapter 10 describes the crystal structure of the substrate-free enzyme. This study shows that no large conformational changes take place upon substrate (analog) binding. The stimulating role of POHB is probably caused by several subtle effects. Stabilisation of the phenolate form of the substrate results in distribution of the electronic charges in the active site. These charge distributions influence the dynamic equilibrium between the "open" and "closed" conformation of FAD in such a way that the nicotinamide ring of NADPH and the isoalloxazine ring of FAD become optimally oriented for efficient reduction.