Thermostable D-amino acid decarboxylases derived from Thermotoga maritima diaminopimelate decarboxylase.

Diaminopimelate decarboxylases (DAPDCs) are highly selective enzymes that catalyze the common final step in different lysine biosynthetic pathways, i.e. the conversion of meso-diaminopimelate (DAP) to L-lysine. We examined the modification of the substrate specificity of the thermostable decarboxylase from Thermotoga maritima with the aim to introduce activity with 2-aminopimelic acid (2-APA) since its decarboxylation leads to 6-aminocaproic acid (6-ACA), a building block for the synthesis of nylon-6. Structure-based mutagenesis of the distal carboxylate binding site resulted in a set of enzyme variants with new activities toward different D-amino acids. One of the mutants (E315T) had lost most of its activity toward DAP and primarily acted as a 2-APA decarboxylase. We next used computational modeling to explain the observed shift in catalytic activities of the mutants. The results suggest that predictive computational protocols can support the redesign of the catalytic properties of this class of decarboxylating PLP-dependent enzymes.

Catalytic and structural properties of ATP-dependent caprolactamase from Pseudomonas jessenii.

Caprolactamase is the first enzyme in the caprolactam degradation pathway of Pseudomonas jessenii. It is composed of two subunits (CapA and CapB) and sequence-related to other ATP-dependent enzymes involved in lactam hydrolysis, like 5-oxoprolinases and hydantoinases. Low sequence similarity also exists with ATP-dependent acetone- and acetophenone carboxylases. The caprolactamase was produced in Escherichia coli, isolated by His-tag affinity chromatography, and subjected to functional and structural studies. Activity toward caprolactam required ATP and was dependent on the presence of bicarbonate in the assay buffer. The hydrolysis product was identified as 6-aminocaproic acid. Quantum mechanical modeling indicated that the hydrolysis of caprolactam was highly disfavored (ΔG0 '= 23 kJ/mol), which explained the ATP dependence. A crystal structure showed that the enzyme exists as an (αß)2 tetramer and revealed an ATP-binding site in CapA and a Zn-coordinating site in CapB. Mutations in the ATP-binding site of CapA (D11A and D295A) significantly reduced product formation. Mutants with substitutions in the metal binding site of CapB (D41A, H99A, D101A, and H124A) were inactive and less thermostable than the wild-type enzyme. These residues proved to be essential for activity and on basis of the experimental findings we propose possible mechanisms for ATP-dependent lactam hydrolysis.

Squalene hopene cyclases are protonases for stereoselective Brønsted acid catalysis.

For many important reactions catalyzed in chemical laboratories, the corresponding enzymes are missing, representing a restriction in biocatalysis. Although nature provides highly developed machineries appropriate to catalyze such reactions, their potential is often ignored. This also applies to Brønsted acid catalysis, a powerful method to promote a myriad of chemical transformations. Here, we report on the unique protonation machinery of a squalene hopene cyclase (SHC). Active site engineering of this highly evolvable enzyme yielded a platform for enzymatic Brønsted acid catalysis in water. This is illustrated by activation of different functional groups (alkenes, epoxides and carbonyls), enabling the highly stereoselective syntheses of various cyclohexanoids while uncoupling SHC from polycyclization chemistry. This work highlights the potential of systematic investigation on nature's catalytic machineries to generate unique catalysts.

Slippery substrates impair function of a bacterial protease ATPase by unbalancing translocation versus exit.

BACKGROUND: ATP-dependent proteases translocate and unfold their substrates. RESULTS: A human virus sequence with only Gly and Ala residues causes similar dysfunctions of eukaryotic and prokaryotic protease motors: unfolding failure. CONCLUSION: Sequences with amino acids of simple shape and small size impair unfolding of contiguous stable domains. SIGNIFICANCE: Compartmented ATP-dependent proteases of diverse origin share conserved principles of interaction between translocase/effector and substrate/recipient. ATP-dependent proteases engage, translocate, and unfold substrate proteins. A sequence with only Gly and Ala residues (glycine-alanine repeat; GAr) encoded by the Epstein-Barr virus of humans inhibits eukaryotic proteasome activity. It causes the ATPase translocase to slip on its protein track, stalling unfolding and interrupting degradation. The bacterial protease ClpXP is structurally simpler than the proteasome but has related elements: a regulatory ATPase complex (ClpX) and associated proteolytic chamber (ClpP). In this study, GAr sequences were found to impair ClpXP function much as in proteasomes. Stalling depended on interaction between a GAr and a suitably spaced and positioned folded domain resistant to mechanical unfolding. Persistent unfolding failure results in the interruption of degradation and the production of partial degradation products that include the resistant domain. The capacity of various sequences to cause unfolding failure was investigated. Among those tested, a GAr was most effective, implying that viral selection had optimized processivity failure. More generally, amino acids of simple shape and small size promoted unfolding failure. The ClpX ATPase is a homohexamer. Partial degradation products could exit the complex through transient gaps between the ClpX monomers or, alternatively, by backing out. Production of intermediates by diverse topological forms of the hexamer was shown to be similar, excluding lateral escape. In principle, a GAr could interrupt degradation because 1) the translocase thrusts forward less effectively or because 2) the translocase retains substrate less well when resetting between forward strokes. Kinetic analysis showed that the predominant effect was through the second of these mechanisms.