Importance of Loop L1 Dynamics for Substrate Capture and Catalysis in Pseudomonas aeruginosa d-Arginine Dehydrogenase

Abstract

Mobile loops located at the active site entrance in enzymes often participate in conformational changes required to shield the reaction from bulk solvent, to control the access of the substrate to the active site, and to position residues for substrate binding and catalysis. In d-arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH), previous crystallographic data suggested that residues 45-47 in the FAD-binding domain and residues 50-56 in the substrate-binding domain in loop L1 could adopt two distinct conformations. In this study, we have used molecular dynamics, kinetics, and fluorescence spectroscopy on the S45A and A46G enzyme variants of PaDADH to investigate the impact of mutations in loop L1 on the catalytic function of the enzyme. Molecular dynamics showed that the mutant enzymes have probabilities of being in open conformations that are higher than that of wild-type PaDADH of loop L1, yielding an increased level of solvent exposure of the active site. In agreement, the flavin fluorescence intensity was ∼2-fold higher in the S45A and A46G enzymes than in wild-type PaDADH, with a 9 nm bathochromic shift of the emission band. In the variant enzymes, the k/K values with d-arginine were ∼13-fold lower than in wild-type PaDADH. Moreover, the pH profiles for the k value with d-arginine showed a hollow, consistent with restricted proton movements in catalysis, and no saturation was achieved with the alternate substrate d-leucine in the reductive half-reaction of the variant enzymes. Taken together, the computational and experimental data are consistent with the dynamics of loop L1 being important for substrate capture and catalysis in PaDADH.

Redox control of iodotyrosine deiodinase

“…33 Active site lid and loop sequences are also known to facilitate substrate capture and sequestration for efficient Fl-dependent catalysis. 34,35 Closure of the active site lid of IYD is distinct from most examples since the conformational change does not accelerate an intrinsic reaction of Fl but rather switches the redox processes available to Fl. The dehalogenation activity of IYD depends on the presence of the full halotyrosine substrate.…”

Conformational Dynamics‐Guided Loop Engineering of an Alcohol Dehydrogenase: Capture, Turnover and Enantioselective Transformation of Difficult‐to‐Reduce Ketones

“…Loop motion and flexibility at the binding pocket play crucial roles in the optimal functions of enzymes. [8d, [9][10][11][12][13][14] Induced fluctuation by introducing appropriate mutations lead to loop movements that can reshape the binding pocket, and therefore influence substrate capture and turnover. To exploit this hypothesis in the present study, the thermostable alcohol dehydrogenase TbSADH was chosen as the model enzyme in asymmetric reduction of ketone 1 that is not accepted by the WT.…”

“…In fact, previous studies have demonstrated that performing site-specific mutagenesis on either single or combined residues lining the binding pocket can efficiently affect the enantioselectivity, substrate specificity and other properties of TbSADH. [8] In recent years, there has been growing interest in the conformational dynamics of proteins, which play an important role in enzyme catalysis, and engineering of enzymes guided by conformational dynamics has become an effective strategy for protein evolution, achieving successes in expanding substrate scope, [9] increasing enantioselectivity, [10] relieving product inhibition, [11] and improving thermostability. [12] The motion of amino acid residues in loops involved in the pocket has been recognized to influence the catalytic properties of enzymes.…”

“…[12] The motion of amino acid residues in loops involved in the pocket has been recognized to influence the catalytic properties of enzymes. [9,13] Although the residues in loops generally do not act as catalytic sites, they often participate in conformational changes required for fine-tuning residues that are relevant to substrate binding and catalysis. [14] Moreover, conformational dynamics influencing the substrate scope of ADHs were explored in previous studies, [6b,8d] which inspires us to extend this subject to the difficult-to-perform asymmetric reduction of bulky ketones such as 1 with formation of (S)-2 (Scheme 1).…”

“…In recent years, there has been growing interest in the conformational dynamics of proteins, which play an important role in enzyme catalysis, and engineering of enzymes guided by conformational dynamics has become an effective strategy for protein evolution, achieving successes in expanding substrate scope, increasing enantioselectivity, relieving product inhibition, and improving thermostability . The motion of amino acid residues in loops involved in the pocket has been recognized to influence the catalytic properties of enzymes . Although the residues in loops generally do not act as catalytic sites, they often participate in conformational changes required for fine‐tuning residues that are relevant to substrate binding and catalysis…”