Escherichia coli amino acid activating enzyme

Few microbial proteases acting on n-peptides are known. The alkaline D-peptidase (ADP) from Bacillus cereus is related to Du-carboxypeptidase and p-lactamases. These enz unes have an accessible groove in which the nucleophilic serine and other catalytic amino acids are located. This n-peptidase could be applied for the synthesis of the 92-amino acid peptidyl prolyl cis-trans isomerase from Escherichia coli by condensation of two peptide fragments, of which the 35-amino acid acyl donor was activated as the OGp ester [62]. Thus the D-amino acid-selective enzyme was used for preparing a protein composed of L-amino acids and making the product insensitive to hydrolysis by the coupling enzyme. 

Hydantoinase-Carbamoylase System for t-Amino Acid Synthesis Despite a number of reports of strains with L-selechve hydantoin-hydrolyzing enzymes [38] the commercial application of the hydantoinase process is stiU restricted to the production of D-amino acids. Processes for the production of L-amino acids are Umited by low space-time yields and high biocatalyst costs. Recently, a new generation of an L-hydantoinase process was developed based on a tailor-made recombinant whole cell biocatalyst. Further reduction of biocatalyst cost by use of recombinant Escherichia coli cells overexpressing hydantoinase, carbamoylase, and hydantoin racemase from Arthrohacter sp. DSM 9771 were achieved. To improve the hydan-toin-converting pathway, the level of expression of the different genes was balanced on the basis of their specific activities. The system has been appUed to the preparation of L-methionine the space-time yield is however still Umited [39]. Improvements in the deracemization process from rac-5-substituted hydantoins to L-amino acids still requires a more selective L-hydantoinase. 

The enzyme is calcium-dependent, has a pH optimum of 8-10, and shows a striking preference for substrate presented in the form of Escherichia coli membranes. Using oligonucleotide probes based on amino-terminal sequence data, the corresponding human gene from a genomic DNA library, has been cloned and expressed in animal cells. The protein is secreted from cells in an active form. The deduced amino acid sequence of the human protein consists of 124 amino acids, contains structural features common to all known PLAjS, and has a half-cysteine pattern that is characteristic of the snake venom group II enzyme (60). 

A unique biochemical target in the HIV-1 replication cycle was revealed when HIV protease was cloned and expressed " in Escherichia coli. HIV protease is an enzyme that cleaves gag-pro propeptides to yield active enzymes that function in the maturation and propagation of new virus. The catalytically active protca.se is a. symmetric dimer of two identical 99 amino acid subunits, each contributing the triad Asp-Thr-Gly to the active site." The homodimer is unlike monomeric asparlyl protea.ses (renin, pepsin, cathep-sin D). which also have different. substrate specificities. The designs of. some inhibitors for HIV-1 protease exploit the C2 symmetry of the enzyme. HIV-1 protease has active site speclnc ity for the triad Tyr-Phe-Pro in the unit Ser-(Thr)-Xaa-Xaa-Tyr-Phc-Pm. whenr Xaa is an arbitrary amino acid. 

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