Dissecting the low catalytic capability of flavin-dependent halogenases

Although flavin-dependent halogenases (FDHs) are attractive biocatalysts, their practical applications are limited because of their low catalytic efficiency. Here, we investigated the reaction mechanisms and structures of tryptophan 6-halogenase (Thal) from Streptomyces albogriseolus using stopped-flow, rapid-quench flow, quantum/mechanics molecular mechanics calculations, crystallography, and detection of intermediate (hypohalous acid [HOX]) liberation. We found that the key flavin intermediate, C4a-hydroperoxyflavin (C4aOOH-FAD), formed by Thal and other FDHs (tryptophan 7-halogenase [PrnA] and tryptophan 5-halogenase [PyrH]), can react with I-, Br-, and Cl- but not F- to form C4a-hydroxyflavin and HOX. Our experiments revealed that I- reacts with C4aOOH-FAD the fastest with the lowest energy barrier and have shown for the first time that a significant amount of the HOX formed leaks out as free HOX. This leakage is probably a major cause of low product coupling ratios in all FDHs. Site-saturation mutagenesis of Lys79 showed that changing Lys79 to any other amino acid resulted in an inactive enzyme. However, the levels of liberated HOX of these variants are all similar, implying that Lys79 probably does not form a chloramine or bromamine intermediate as previously proposed. Computational calculations revealed that Lys79 has an abnormally lower pKa compared with other Lys residues, implying that the catalytic Lys may act as a proton donor in catalysis. Analysis of new X-ray structures of Thal also explains why premixing of FDHs with reduced flavin adenine dinucleotide generally results in abolishment of C4aOOH-FAD formation. These findings reveal the hidden factors restricting FDHs capability which should be useful for future development of FDHs applications.</p

The nucleation of monomeric parallel beta-sheet-like structures and their self-assembly in aqueous solution

The aromatic diacid residue 4,6-dibenzofuranbispropionic acid (1) was designed to nucleate a parallel beta-sheet-like structure in small peptides in aqueous solution via a hydrogen-bonded hydrophobic cluster. Even though a 14-membered ring hydrogen bond necessary for parallel beta-sheet formation is favored in simple amides composed of 1, this hydrogen bonding interaction does not appear to be sufficient to nucleate parallel beta-sheet formation in the absence of hydrophobic clustering between the dibenzofuran portion of 1 and the hydrophobic side chains of the flanking alpha-amino acids. The subsequence --hydrophobic residue-1-hydrophobic residue-- is required for folding in the context of a nucleated two-stranded parallel beta-sheet structure. In all cases where the peptidomimetics can fold into two diastereomeric parallel beta-sheet structures having different hydrogen bonding networks, these conformations appear to exchange rapidly. The majority of the parallel beta-sheet structures evaluated herein undergo linked intramolecular folding and self-assembly, affording a fibrillar beta-sheet quaternary structure. To unlink folding and assembly, asymmetric parallel beta-sheet structures incorporating N-methylated alpha-amino acid residues have been synthesized using a new solid phase approach. Residue 1 facilitates the folding of several peptides described within affording a monomeric parallel beta-sheet-like structure in aqueous solution, as ascertained by a variety of spectroscopic and biophysical methods, increasing our understanding of parallel beta-sheet structure

Improved X‐ray diffraction from Bacillus megaterium penicillin G acylase crystals through long cryosoaking dehydration

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/89569/1/S1744309111040462.pd

Crystallization and preliminary crystallographic studies of dihydrofolate reductase-thymidylate synthase from Trypanosoma cruzi, the Chagas disease pathogen

Crystals of complexes of the T. cruzi dihydrofolate reductase-thymidylate synthase enzyme with three antifolates in two space groups have been obtained that diffracted to 2.1–2.8 Å resolution. The antifolates used for cocrystallization were dihydrotriazine-based and quinazoline-based antifolates