Electrochemical and Structural Study of the Buried Tryptophan in Azurin: Effects of Hydration and Polarity on the Redox Potential of W48.

Tryptophan serves as an important redox-active amino acid in mediating electron transfer and mitigating oxidative damage in proteins. We previously showed a difference in electrochemical potentials for two tryptophan residues in azurin with distinct hydrogen-bonding environments. Here, we test whether reducing the side chain bulk at position Phe110 to Leu, Ser, or Ala impacts the electrochemical potentials (E°) for tryptophan at position 48. X-ray diffraction confirmed the influx of crystallographically resolved water molecules for both the F110A and F110L tyrosine free azurin mutants. The local environments of W48 in all azurin mutants were further evaluated by UV resonance Raman (UVRR) spectroscopy to probe the impact of mutations on hydrogen bonding and polarity. A correlation between the frequency of the ω17 mode─considered a vibrational marker for hydrogen bonding─and E° is proposed. However, the trend is opposite to the expectation from a previous study on small molecules. Density functional theory calculations suggest that the ω17 mode reflects hydrogen bonding as well as local polarity. Further, the UVRR data reveal different intensity/frequency shifts of the ω9/ω10 vibrational modes that characterize the local H-bonding environments of tryptophan. The cumulative data support that the presence of water increases E° and reveal properties of the protein microenvironment surrounding tryptophan.

Crystal structures of non-oxidative decarboxylases reveal a new mechanism of action with a catalytic dyad and structural twists.

Hydroxybenzoic acids, like gallic acid and protocatechuic acid, are highly abundant natural compounds. In biotechnology, they serve as critical precursors for various molecules in heterologous production pathways, but a major bottleneck is these acids' non-oxidative decarboxylation to hydroxybenzenes. Optimizing this step by pathway and enzyme engineering is tedious, partly because of the complicating cofactor dependencies of the commonly used prFMN-dependent decarboxylases. Here, we report the crystal structures (1.5-1.9 Å) of two homologous fungal decarboxylases, AGDC1 from Arxula adenivorans, and PPP2 from Madurella mycetomatis. Remarkably, both decarboxylases are cofactor independent and are superior to prFMN-dependent decarboxylases when heterologously expressed in Saccharomyces cerevisiae. The organization of their active site, together with mutational studies, suggests a novel decarboxylation mechanism that combines acid-base catalysis and transition state stabilization. Both enzymes are trimers, with a central potassium binding site. In each monomer, potassium introduces a local twist in a ß-sheet close to the active site, which primes the critical H86-D40 dyad for catalysis. A conserved pair of tryptophans, W35 and W61, acts like a clamp that destabilizes the substrate by twisting its carboxyl group relative to the phenol moiety. These findings reveal AGDC1 and PPP2 as founding members of a so far overlooked group of cofactor independent decarboxylases and suggest strategies to engineer their unique chemistry for a wide variety of biotechnological applications.

Solution Structure and Conformational Flexibility of a Polyketide Synthase Module.

Polyketide synthases (PKSs) are versatile C-C bond-forming enzymes that are broadly distributed in bacteria and fungi. The polyketide compound family includes many clinically useful drugs such as the antibiotic erythromycin, the antineoplastic epothilone, and the cholesterol-lowering lovastatin. Harnessing PKSs for custom compound synthesis remains an open challenge, largely because of the lack of knowledge about key structural properties. Particularly, the domains-well characterized on their own-are poorly understood in their arrangement, conformational dynamics, and interplay in the intricate quaternary structure of modular PKSs. Here, we characterize module 2 from the 6-deoxyerythronolide B synthase by small-angle X-ray scattering and cross-linking mass spectrometry with coarse-grained structural modeling. The results of this hybrid approach shed light on the solution structure of a cis-AT type PKS module as well as its inherent conformational dynamics. Supported by a directed evolution approach, we also find that acyl carrier protein (ACP)-mediated substrate shuttling appears to be steered by a nonspecific electrostatic interaction network.