Redesigning the PheA Domain of Gramicidin Synthetase Leads to a New Understanding of the Enzyme's Mechanism and Selectivity

Abstract

The PheA domain of gramicidin synthetase A, a non-ribosomal peptide synthetase, selectively binds phenylalanine along with ATP and Mg2+ and catalyzes the formation of an aminoacyl adenylate. In this study, we have used a novel protein redesign algorithm, K*, to predict mutations in PheA that should exhibit improved binding for tyrosine. Interestingly, the introduction of two predicted mutations to PheA did not significantly improve KD, as measured by equilibrium fluorescence quenching. However, the mutations improved the specificity of the enzyme for tyrosine (as measured by kcat/KM), primarily driven by a 56-fold improvement in KM, although the improvement did not make tyrosine the preferred substrate over phenylalanine. Using stopped-flow fluorometry, we examined binding of different amino acid substrates to the wild-type and mutant enzymes in the pre-steady state in order to understand the improvement in KM. Through these investigations, it became evident that substrate binding to the wild-type enzyme is more complex than previously described. These experiments show that the wild-type enzyme binds phenylalanine in a kinetically selective manner; no other amino acids tested appeared to bind the enzyme in the early time frame examined (500 ms). Furthermore, experiments with PheA, phenylalanine, and ATP reveal a two-step binding process, suggesting that the PheA-ATP-phenylalanine complex may undergo a conformational change toward a catalytically relevant intermediate on the pathway to adenylation; experiments with PheA, phenylalanine, and other nucleotides exhibit only a one-step binding process. The improvement in KM for the mutant enzyme toward tyrosine, as predicted by K*, may indicate that redesigning the side-chain binding pocket allows the substrate backbone to adopt productive conformations for catalysis but that further improvements may be afforded by modeling an enzyme:ATP:substrate complex, which is capable of undergoing conformational change.

Computational structure-based redesign of enzyme activity

“…Luo et al (30) claimed that the discrimination of the amino acid substrate begins when the transition state is formed during the catalysis. Stevens et al (3) suggested that a conformational change toward a catalytically relevant intermediate occurs in the adenylation process of PheA. In our results, the double mutant T278L/A301G dramatically lowers the value of K m for the noncognate amino acid Leu from the WT PheA, requiring changes to only two residues in the active site.…”

“…Several studies have shown that the substrate specificity of the adenylation domain can be modified by the mutation of the active-site residues (3,24,31). By using a multiple sequence alignment approach to redesign the substrate specificity of GrsA-PheA, Stachelhaus et al (24) successfully improved the activity of the enzyme for the noncognate amino acid Leu with the introduction of a double mutation, T278M/A301G, and altered the substrate specificity of an aspartate-activating domain AspA to Asn by a single mutation H322E in the active site.…”

“…Plasmid pQE60 containing WT and A301G mutant PheA genes from Bacillus brevis (GI: 39366) were obtained as described in ref. 3. Mutagenesis of mutant PheA.…”

BWM*: A Novel, Provable, Ensemble-Based Dynamic Programming Algorithm for Sparse Approximations of Computational Protein Design

“…Several protein design algorithms have successfully predicted protein sequences that fold and bind the desired target in vitro (Frey et al, 2010;Roberts et al, 2012;Rudicell et al, 2014;Stevens et al, 2006;Georgiev et al, 2012;Georgiev and Donald, 2007;Georgiev et al, 2014;Donald, 2011), and even in vivo (Reeve et al, 2015;Roberts et al, 2012;Rudicell et al, 2014;Georgiev et al, 2012;Georgiev et al, 2014;Donald, 2011). However, protein design is NP-hard (Kingsford et al, 2005), making algorithms that guarantee optimality expensive for larger designs where many residues are allowed to mutate simultaneously.…”

“…By explicitly modeling proteins as a thermodynamic ensemble of molecular conformations, osprey/K* has successfully designed sequences that have performed well both in vitro Chen et al, 2009;Frey et al, 2010;Rudicell et al, 2014;Georgiev et al, 2012;Gorczynski et al, 2007;Stevens et al, 2006) and in vivo Gorczynski et al, 2007;Rudicell et al, 2014;Frey et al, 2010), as well as in non-human primates . K* accomplishes this by using dead end elimination followed by A* (DEE/A*) (Leach and Lemon, 1998;Goldstein, 1994) to provably compute a gap-free list of conformations within an energy window E w of the GMEC, and provably approximate partition functions over molecular ensembles.…”