[NiFe]‐hydrogenase (Hyd) 2 of Escherichia coli has been proposed to generate proton motive force during H2‐oxidation, which it is dependent on if cells are incubated anaerobically with glycerol to drive reverse H2‐production. The integral membrane subunit HybB is required for proton transfer (PT) by Hyd‐2 but has no cofactor. To provide evidence for PT by HybB, we analyzed the roles of conserved amino acid residues in a predicted proton channel. Exchange of conserved residues identified residues Y99, E133, H184, and E228 as mandatory for PT from the cytoplasm and quinol oxidation. In contrast, exchange of W54, D58, or R89 rendered Hyd‐2 uni‐directional and influenced the equilibrium. Our findings show that HybB is the key subunit in PT.
Trypsin is a long-known serine protease widely used in biochemical, analytical, biotechnological, or biocatalytic applications. The high biotechnological potential is based on its high catalytic activity, substrate specificity, and catalytic robustness in non-physiological reaction conditions. The latter is mainly due to its stable protein fold, to which six intramolecular disulfide bridges make a significant contribution. Although trypsin does not depend on cofactors, it essentially requires the binding of calcium ions to its calcium-binding site to obtain complete enzymatic activity and stability. This behavior is inevitably associated with a limitation of the enzyme’s applicability. To make trypsin intrinsically calcium-independent, we removed the native calcium-binding site and replaced it with another disulfide bridge. The resulting stabilized apo-trypsin (aTn) retains full catalytic activity as proven by enzyme kinetics. Studies using Ellmann’s reagent further prove that the two inserted cysteines at positions Glu70 and Glu80 are in their oxidized state, creating the desired functional disulfide bond. Furthermore, aTn is independent of calcium ions, possesses increased thermal and functional stability, and significantly reduced autolysis compared to wildtype trypsin. Finally, we confirmed our experimental data by solving the X-ray crystal structure of aTn.
The application of D -stereospecific proteases ( D SPs) in resolution of racemic amino acids and in the semisynthesis of proteins has been a successful strategy. The main limitation for a broader application is, however, the accessibility of suitable D SPs covering multiple substrate specificities. To identify D SPs with novel primary substrate preferences, a fast specificity screening method using the easily accessible internally quenched fluorogenic substrate aminobenzoyl- D -arginyl- D -alanyl- p -nitroanilide was developed. By monitoring both UV/ vis -absorbance and fluorescence signals at the same time it allows to detect two distinct D -amino acid substrate specificities simultaneously and separately with respect to the individual specificities. In order to identify novel D SP specificities for synthesis applications, D SPs specific for D -arginine were of special interest due to their potential ability as catalysts for substrate mimetics-mediated peptide and protein ligations. D -alanine in the substrate served as positive control and reference based on its known acceptance by numerous D SPs. In silico analysis suggested that D SPs are predominantly present in gram-positive microorganisms, therefore this study focused on the bacilli strains Bacillus thuringiensis and Bacillus subtilis as potential hosts of D -Arg-specific D SPs. While protease activities toward D -alanine were found in both organisms, a novel and so far unknown D -arginine specific D SP was detected within the culture supernatant of B. thuringiensis . Enrichment of this activity via cation exchange and size exclusion chromatography allowed isolation and further characterization of this novel enzyme consisting of a molecular mass of 37.7 kDa and an enzymatic activity of 8.3 U mg -1 for cleaving the D -Arg| D -Ala bond in the detecting substrate. Independent experiments also showed that the identified enzyme shows similarities to the class of penicillin binding proteins. In future applications this enzyme will be a promising starting point for the development of novel strategies for the semisynthesis of all - L -proteins.
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