Direct coordination of pterin to FeII enables neurotransmitter biosynthesis in the pterin-dependent hydroxylases.

The pterin-dependent nonheme iron enzymes hydroxylate aromatic amino acids to perform the biosynthesis of neurotransmitters to maintain proper brain function. These enzymes activate oxygen using a pterin cofactor and an aromatic amino acid substrate bound to the FeII active site to form a highly reactive FeIV = O species that initiates substrate oxidation. In this study, using tryptophan hydroxylase, we have kinetically generated a pre-FeIV = O intermediate and characterized its structure as a FeII-peroxy-pterin species using absorption, Mössbauer, resonance Raman, and nuclear resonance vibrational spectroscopies. From parallel characterization of the pterin cofactor and tryptophan substrate-bound ternary FeII active site before the O2 reaction (including magnetic circular dichroism spectroscopy), these studies both experimentally define the mechanism of FeIV = O formation and demonstrate that the carbonyl functional group on the pterin is directly coordinated to the FeII site in both the ternary complex and the peroxo intermediate. Reaction coordinate calculations predict a 14 kcal/mol reduction in the oxygen activation barrier due to the direct binding of the pterin carbonyl to the FeII site, as this interaction provides an orbital pathway for efficient electron transfer from the pterin cofactor to the iron center. This direct coordination of the pterin cofactor enables the biological function of the pterin-dependent hydroxylases and demonstrates a unified mechanism for oxygen activation by the cofactor-dependent nonheme iron enzymes.

Computing Cellulase Kinetics with a Two-Domain Linear Interaction Energy Approach.

While heterogeneous enzyme reactions play an essential role in both nature and green industries, computational predictions of their catalytic properties remain scarce. Recent experimental work demonstrated the applicability of the Sabatier principle for heterogeneous biocatalysis. This provides a simple relationship between binding strength and the catalytic rate and potentially opens a new way for inexpensive computational determination of kinetic parameters. However, broader implementation of this approach will require fast and reliable prediction of binding free energies of complex two-phase systems, and computational procedures for this are still elusive. Here, we propose a new framework for the assessment of the binding strengths of multidomain proteins, in general, and interfacial enzymes, in particular, based on an extended linear interaction energy (LIE) method. This two-domain LIE (2D-LIE) approach was successfully applied to predict binding and activation free energies of a diverse set of cellulases and resulted in robust models with high accuracy. Overall, our method provides a fast computational screening tool for cellulases that have not been experimentally characterized, and we posit that it may also be applicable to other heterogeneously acting biocatalysts.

Isoform-Specific Substrate Inhibition Mechanism of Human Tryptophan Hydroxylase.

Tryptophan hydroxylase (TPH) catalyzes the initial and rate-limiting step in the biosynthesis of serotonin, which is associated with a variety of disorders such as depression and irritable bowel syndrome. TPH exists in two isoforms: TPH1 and TPH2. TPH1 catalyzes the initial step in the synthesis of serotonin in the peripheral tissues, while TPH2 catalyzes this step in the brain. In this study, the steady-state kinetic mechanism for the catalytic domain of human TPH1 has been determined. Varying substrate tryptophan (Trp) and tetrahydrobiopterin (BH4) results in a hybrid Ping Pong-ordered mechanism in which the reaction can either occur through a Ping Pong or a sequential mechanism depending on the concentration of tryptophan. The catalytic domain of TPH1 shares a sequence identity of 81% with TPH2. Despite the high sequence identity, differences in the kinetic parameters of the isoforms have been identified; i.e., only TPH1 displays substrate tryptophan inhibition. This study demonstrates that the difference can be traced to an active site loop which displays different properties in the TPH isoforms. Steady-state kinetic results of the isoforms, and variants with point mutations in a loop lining the active site, show that the kinetic parameters of only TPH1 are significantly changed upon mutations. Mutations in the active site loop of TPH1 result in an increase in the substrate inhibition constant, Ki, and therefore turnover rate. Molecular dynamics simulations reveal that this substrate inhibition mechanism occurs through a closure of the cosubstrate, BH4, binding pocket, which is induced by Trp binding.

Stabilization of tryptophan hydroxylase 2 by l-phenylalanine-induced dimerization.

Tryptophan hydroxylase 2 (TPH2) catalyses the initial and rate-limiting step in the biosynthesis of serotonin, which is associated with a variety of disorders such as depression, obsessive compulsive disorder, and schizophrenia. Full-length TPH2 is poorly characterized due to low purification quantities caused by its inherent instability. Three truncated variants of human TPH2 (rch TPH2; regulatory and catalytic domain, NΔ47-rch TPH2; truncation of 47 residues in the N terminus of rch TPH2, and ch TPH2; catalytic domain) were expressed, purified, and examined for changes in transition temperature, inactivation rate, and oligomeric state. ch TPH2 displayed 14- and 11-fold higher half-lives compared to rch TPH2 and NΔ47-rch TPH2, respectively. Differential scanning calorimetry experiments demonstrated that this is caused by premature unfolding of the less stable regulatory domain. By differential scanning fluorimetry, the unfolding transitions of rch TPH2 and NΔ47-rch TPH2 are found to shift from polyphasic to apparent two-state by the addition of l-Trp or l-Phe. Analytical gel filtration revealed that rch TPH2 and NΔ47-rch TPH2 reside in a monomer-dimer equilibrium which is significantly shifted toward dimer in the presence of l-Phe. The dimerizing effect induced by l-Phe is accompanied by a stabilizing effect, which resulted in a threefold increase in half-lives of rch TPH2 and NΔ47-rch TPH2. Addition of l-Phe to the purification buffer significantly increases the purification yields, which will facilitate characterization of hTPH2.

Computational investigation of enthalpy-entropy compensation in complexation of glycoconjugated bile salts with ß-cyclodextrin and analogs.

The inclusion complexes of glycoconjugated bile salts with ß-cyclodextrin (ß-CD) and 2-hydroxypropyl-ß-cyclodextrins (HP-ß-CD) in aqueous solution were investigated by molecular dynamics simulations to provide a molecular explanation of the experimentally observed destabilizing effect of the HP substituents. Good agreement with experimental data was found with respect to penetration depths of CDs. An increased degree of HP substitution (DS) resulted in an increased probability of blocking the cavity opening, thereby hindering the bile salt from entering CD. Further, the residence time of water molecules in the cavity increased with the DS. Release of water from the cavity resulted in a positive enthalpy change, which correlates qualitatively with the experimentally determined increase in complexation enthalpy and contributes to the enthalpy-entropy compensation. The positive change in complexation entropy with DS was not able to compensate for this unfavorable change in enthalpy induced by the HP substituents, resulting in a destabilizing effect. This was found to originate from fixation of the HP substituents and decreased free rotation of the bile salts within the CD cavities.