The crystal structures of three amidohydrolases have been determined recently: glutamine PRPP amidotransferase (GAT), penicillin acylase, and the proteasome. These enzymes use the side chain of the amino-terminal residue, incorporated in a beta-sheet, as the nucleophile in the catalytic attack at the carbonyl carbon. The nucleophile is cysteine in GAT, serine in penicillin acylase, and threonine in the proteasome. Here we show that all three enzymes share an unusual fold in which the nucleophile and other catalytic groups occupy equivalent sites. This fold provides both the capacity for nucleophilic attack and the possibility of autocatalytic processing. We suggest the name Ntn (N-terminal nucleophile) hydrolases for this structural superfamily of enzymes which appear to be evolutionarily related but which have diverged beyond any recognizable sequence similarity.

A protein catalytic framework with an N-terminal nucleophile is capable of self-activation

Summary
In-frame deletions were introduced into each of the eight genes of the hyc operon coding for products required for the formation of the formate hydrogenlyase (FHL) system. The deletions were transferred to the chromosome and the resulting mutants were analysed for development of formate dehydrogenase H and hydrogenase 1, 2 and 3 activity. It was found that hycA, the promoter-proximal gene, is a regulatory gene and that it codes for a product counteracting transcriptional activation by FhlA. Deletions within the hycB to hycH genes specifically affected formate dehydrogenase H activity or hydrogenase 3 activity, or both. None of the mutations affected hydrogenase 1 or 2 activity. A model is proposed for the functional interaction of the different hyc operon gene products in the formate hydrogenlyase complex, which is based on the results of the mutational analysis, on the determination of the subcellular localization of the FdhF, HycE, HycF and HycG polypeptides and on the similarity of hyc gene product sequences with those from other hydrogenase systems. HycH, the product of the most promoter-distal gene, does not seem to form part of the functional FHL complex but rather is required for the conversion of a precursor form of the large subunit of hydrogenase 3 into the mature form.

Mutational analysis of the operon (hyc) determining hydrogenase 3 formation in Escherichia coli.

Ligand-induced conformational change in penicillin acylase.

Intramolecular autoproteolysis initiates the maturation of penicillin amidase from Escherichia coli.

Cephalosporin acylases are a group of enzymes that hydrolyze cephalosporin C (CPC) and/or glutaryl 7-amino cephalosporanic acid (GL-7ACA) to produce 7-amino cephalosporanic acid (7-ACA). The acylase from Pseudomonas sp. 130 (CA-130) is highly active on GL-7ACA and glutaryl 7-aminodesacetoxycephalosporanic acid (GL-7ADCA), but much less active on CPC and penicillin G. The gene encoding the enzyme is expressed as a precursor polypeptide consisting of a signal peptide followed by α- and β-subunits, which are separated by a spacer peptide. Removing the signal peptide has little effect on precursor processing or enzyme activity. Substitution of the first residue of the β-subunit, Ser, results in a complete loss of enzyme activity, and substitution of the last residue of the spacer, Gly, leads to an inactive and unprocessed precursor. The precursor is supposed to be processed autocatalytically, probably intramolecularly. The two subunits of the acylase, which separately are inactive, can generate enzyme activity when coexpressed in Escherichia coli. Data on this and other related acylases indicate that the cephalosporin acylases may belong to a novel class of enzymes (N-terminal nucleophile hydrolases) described recently.

In vivo post-translational processing and subunit reconstitution of cephalosporin acylase from Pseudomonas sp. 130.

The two constituent subunits of the enzyme penicillin acylase from Escherichia coli strain ATCC 11105 are derived from a single precursor polypeptide by post-translational processing. Mutant penicillin acylase precursors were constructed carrying insertions and deletions in various domains and they were analysed for their processing behaviour. It was found that an endopeptide region of appropriate size and an intact C-terminus were absolutely necessary for the maturation process. Internal deletions within the β-subunit domain also prevented post-translational cleavage. Processing competence, therefore, was not merely determined by the amino acid sequence in the vicinity of the processing sites but relied on a correct overall conformation of the protein. The processing pathway in vivo proceeds via an intermediate comprising the α subunits plus endopeptide and is thus identical to the pathway which has been determined previously by in vitro analysis. The post-translational modification of the precursor is probably not carried out by a specific processing enzyme(s) as the heterologous expression of the penicillin acylase (pac) structural gene yielded processed and active enzyme in different enterobacteria and in a Pseudomonas species.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1990.tb19207.x

Primary structure requirements for the maturation in vivo of penicillin acylase from Escherichia coli ATCC 11105

The enzyme penicillin acylase is synthesized by Escherichia coli only at growth temperatures below 30 degrees C. The biochemical basis of this strict temperature-dependent formation of an enzyme was investigated. When the gene (pac) was under the control of the lacUV5 promoter it showed the same temperature-dependent expression as the chromosomally encoded gene transcribed from its own promoter. This indicates that translation of the pac mRNA rather than transcription of the gene is temperature-dependent. This conclusion could be further confirmed by Northern hybridisation and by analysis of pac-lacZ transcriptional fusions. TnphoA insertion mutagenesis and experiments in which the promoter and 5' sequence encoding the signal peptide of the pac gene was exchanged with those of the cyclodextrin glycosyltransferase gene from Klebsiella oxytoca localised the region of pac mRNA responsible for the temperature-sensitive translation to the 5'-untranslated region and/or the signal peptide. Extension of the 5 nucleotide long spacer separating the Shine-Dalgarno motif from the AUG initiation codon by one or three nucleotides lead to partial or full synthesis of penicillin acylase precursor at 40 degrees C, respectively. The precursor of penicillin acylase formed at 40 degrees C by the mutant variants or when placed under the control of a heterologous upstream region was associated with the membrane but could not be translocated. Taken together these data suggest that transport and translation of the penicillin acylase precursor are coupled and that the short Shine-Dalgarno-AUG distance interferes with a competent interaction between the translation initiation complex and the export system at high temperature. Moreover, evidence was also provided which indicates a direct effect of temperature on the conformation of the precursor and it is proposed that the lack of translation at high temperatures has been selected to prevent the accumulation of transport-incompetent protein locked in the membrane.

Corinna Keilmann

Papers

Primary structure requirements for the maturation in vivo of penicillin acylase from Escherichia coli ATCC 11105

Molecular basis of the exclusive low-temperature synthesis of an enzyme in E. coli: penicillin acylase.