Proteins are intrinsically flexible macromolecules that undergo internal motions with time scales spanning femtoseconds to milliseconds. These fluctuations are implicated in the optimization of reaction barriers for enzyme catalyzed reactions. Time, temperature, and mutation dependent hydrogen−deuterium exchange coupled to mass spectrometry (HDX-MS) has been previously employed to identify spatially resolved, catalysis-linked dynamical regions of enzymes. We now extend this technique to pursue the correlation of protein flexibility and chemical reactivity within the diverse and widespread TIM barrel proteins, targeting murine adenosine deaminase (mADA) that catalyzes the irreversible deamination of adenosine to inosine and ammonia. Following a structure−function analysis of rate and activation energy for a series of mutations at a second sphere phenylalanine positioned in proximity to the bound substrate, the catalytically impaired Phe61Ala with an elevated activation energy (Ea = 7.5 kcal/mol) and the wild type (WT) mADA (Ea = 5.0 kcal/mol) were selected for HDX-MS experiments. The rate constants and activation energies of HDX for peptide segments are quantified and used to assess mutation-dependent changes in local and distal motions. Analyses reveal that approximately 50% of the protein sequence of Phe61Ala displays significant changes in the temperature dependence of HDX behaviors, with the dominant change being an increase in protein flexibility. Utilizing Phe61Ile, which displays the same activation energy for k cat as WT, as a control, we were able to further refine the HDX analysis, highlighting the regions of mADA that are altered in a functionally relevant manner. A map is constructed that illustrates the regions of protein that are proposed to be essential for the thermal optimization of active site configurations that dominate reaction barrier crossings in the native enzyme.

Human catechol O-methyltransferase (COMT) has emerged as a model for understanding enzyme-catalyzed methyl transfer from S-adenosylmethionine (AdoMet) to small-molecule catecholate acceptors. Mutation of a single residue (tyrosine 68) behind the methyl-bearing sulfonium of AdoMet was previously shown to impair COMT activity by interfering with methyl donor–acceptor compaction within the activated ground state of the wild type enzyme [J. Zhang, H. J. Kulik, T. J. Martinez, J. P. Klinman, Proc. Natl. Acad. Sci. U.S.A. 112, 7954–7959 (2015)]. This predicts the involvement of spatially defined protein dynamical effects that further tune the donor/acceptor distance and geometry as well as the electrostatics of the reactants. Here, we present a hydrogen/deuterium exchange (HDX)-mass spectrometric study of wild type and mutant COMT, comparing temperature dependences of HDX against corresponding kinetic and cofactor binding parameters. The data show that the impaired Tyr68Ala mutant displays similar breaks in Arrhenius plots of both kinetic and HDX properties that are absent in the wild type enzyme. The spatial resolution of HDX below a break point of 15–20 °C indicates changes in flexibility across ∼40% of the protein structure that is confined primarily to the periphery of the AdoMet binding site. Above 20 °C, Tyr68Ala behaves more like WT in HDX, but its rate and enthalpic barrier remain significantly altered. The impairment of catalysis by Tyr68Ala can be understood in the context of a mutationally induced alteration in protein motions that becomes manifest along and perpendicular to the primary group transfer coordinate.

4-Hydroxyphenylacetate 3-hydroxylase (EcHpaB) from Escherichia coli is capable of efficient ortho -hydroxylation of a wide range of phenolic compounds and demonstrates great potential for broad chemoenzymatic applications. To understand the structural and mechanistic basis of its catalytic versatility, we elucidated the crystal structure of EcHpaB by X-ray crystallography, which revealed a unique loop structure covering the active site. We further performed mutagenesis studies of this loop to probe its role in substrate specificity and catalytic activity. Our results not only showed the loop has great plasticity and strong tolerance towards extensive mutagenesis, but also suggested a flexible loop that enables the entrance and stable binding of substrates into the active site is the key factor to the enzyme catalytic versatility. These findings lay the groundwork for editing the loop sequence and structure for generation of EcHpaB mutants with improved performance for broader laboratory and industrial use.

2-Azabicyclo[2.2.1]hept-5-en-3-one (γ-lactam) is an important precursor of many carbocyclic nucleoside analogs and pharmaceuticals. (-)-γ-Lactam has attracted much attention because of its role as an intermediate of antiviral drugs such as abacavir and carbovir. (+)-γ-Lactamase can be used for the kinetic resolution of γ-lactam to obtain (-)-γ-lactam. In this study, a novel (+)-γ-lactamase (Mh33H4-5540) was discovered from the gene library of Microbacterium hydrocarbonoxydans based on a colorimetric high-throughput screening method and it could be used to enantioselectively catalyze the bioresolution of racemic γ-lactam with high enantiomeric excess (ee) (>99 %) and yield (>49 %). An unexpected finding was that Mh33H4-5540 was unrelated to other known γ-lactamases (5.7, 4.8, 7.2, and 5.4 % similarities in amino sequence with (+)-γ-lactamase from Comamonas acidovorans, Bradyrhizobium japonicum, Aeropyrum pernix, and Sulfolobus solfataricus, respectively) but rather related to isochorismatases. The homolog analysis of Mh33H4-5540 revealed that it was similar in structure with bacterial isochorismatases (an isochorismatase from Pseudomonas putida (PDB number 4H17) and a putative isochorismatase from Oleispira antarctica (PDB number 3LQY)). Thus, Mh33H4-5540 represented another type of (+)-γ-lactamase. Mh33H4-5540 was overexpressed in E. coli Rosetta (DE3), purified to homogeneity and functionally characterized. The enzyme displayed optimal activity at 25 °C and pH 8.0. The activity showed a 5.5-fold increase in the presence of 0.5 M Ni or Co. Mh33H4-5540 displayed much higher (+)-γ-lactamase activity than any other biochemically characterized (+)-γ-lactamases. Overall, we discovered a novel (+)-γ-lactamase Mh33H4-5540 which displayed the highest activity. It could be a promising candidate of biocatalyst for industrial applications of highly valuable chiral pharmaceutical chemicals.

(+)-γ-lactamase catalyzes the specific hydrolysis of (+)-γ-lactam out of the racemic γ-lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one) to leave optically pure (−)-γ-lactam, which is the key building block of antiviral drugs such as carbovir and abacavir. However, no structural data has been reported on how the enzymes bind the γ-lactams and achieve their enantioselectivities. We previously identified an isochorismatase-like hydrolase (IHL, Mh33H4-5540) with (+)-γ-lactamase activity, which constitutes a novel family of γ-lactamase. Here, we first discovered that this enzyme actually hydrolyzed both (+)- and (−)-γ-lactam, but with apparently different specificities. We determined the crystal structures of the apo-form, (+)-γ-lactam bound, and (−)-γ-lactam bound forms of the enzyme. The structures showed that the binding sites of both (+) and (−)-γ-lactam resemble those of IHLs, but the “cover” loop conserved in IHLs is lacking in the enzyme, probably resulting in its incomplete enantioselectivity. Structural, biochemical, and molecular dynamics simulation studies demonstrated that the steric clash caused by the binding-site residues, especially the side-chain of Cys111 would reduce the binding affinity of (−)-γ-lactam and possibly the catalytic efficiency, which might explain the different catalytic specificities of the enantiomers of γ-lactam. Our results would facilitate the directed evolution and application of Mh33H4-5540 in antiviral drug synthesis.

No abstract

No abstract

A novel (؉)-␥-lactamase used for the resolution of racemic ␥-lactam from Bradyrhizobium japonicum USDA 6 was found as a result of sequence-structure guided genome mining. It consists of 409 amino acids, only 49% of which are identical to the amino acid sequences of the known (؉)-␥-lactamase from Sulfolobus solfataricus. This is only the third (؉)-␥-lactamase gene to be reported.