Purification, cloning, and primary structure of a new enantiomer-selective amidase from a Rhodococcus strain: structural evidence for a conserved genetic coupling with nitrile hydratase

Abstract

A new enantiomer-selective amidase active on several 2-aryl propionamides was identified and purified from a newly isolated Rhodococcus strain. The characterized amidase is an apparent homodimer, each molecule of which has an Mr of 48,554; it has a specific activity of 16.5 g,mol of S(+)-2-phenylpropionic acid formed per min per mg of enzyme from the racemic amide under our conditions. An oligonucleotide probe was deduced from limited peptide information and was used to clone the corresponding gene, named amdA. As expected, significant homologies were found between the amino acid sequences of the enantiomer-selective amidase of Rhodococcus sp., the corresponding enzyme from Brevibacterium sp. strain R312, and several known amidases, thus confirming the existence of a structural class of amidase enzymes. Genes probably coding for the two subunits of a nitrite hydratase, albeit in an inverse order, were found 39 bp downstream of amdA, suggesting that such a genetic organization might be conserved in different microorganisms. Although we failed to express an active Rhodococcus amidase in Escherichia coli, even in conditions allowing the expression of an active R312 enzyme, the high-level expression of the active recombinant enzyme could be demonstrated in Brevibacterium lactofermentum by using a pSRl-derived shuttle vector.After a systematic search for soil microorganisms able to carry out the enantiomer-selective hydrolysis of racemic nitriles or amides, especially 2-aryl propionamides, we recently reported the purification and cloning of such an amidolytic activity from Brevibacterium sp. strain R312 (15). We found that the gene coding for the enantiomer-selective amidase, amdA, was closely linked to the genes coding for the two subunits, a and ,B, of nitrile hydratase, the enzyme responsible for the hydration of various nitriles to the corresponding amides (10). The structure of this region led to the hypothesis that the two genes could be translated from a polycistronic mRNA, thus supporting the view that there is a nitrile utilization operon, at least in this bacterium. These results have been confirmed by another group (9). Moreover, it was found that the identified amidase displays a striking homology to other known amidases, such as the acetamidase from Aspergillus nidulans and the indoleacetamide hydrolases from Pseudomonas syringae (savastanoi) and Agrobacterium tumefaciens. Interestingly, the homology was not scattered through the whole amino acid sequence but was mainly focused in a portion of the sequence around amino acids 150 to 220 of the R312 sequence (47 to 65% strict identity), strongly suggesting that this conserved sequence could be part of the active site of this class of enzymes.We now report the identification of a new Rhodococcus strain, obtained during a microbiological screening of soil samples, which is also characterized by efficient stereospecific amidase activity on 2-aryl propionamides. The enzyme responsible for this activity was purified, cloned, and sequenced. It is significantly ...

Glu-tRNA Gln amidotransferase: A novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation

“…On the other hand, there is significant amino acid sequence similarity (over the entire protein, see Fig. 6B) of the A subunit of B. subtilis Glu-AdT with an amidase from the Gram-positive organism Rhodococcus (26). The amidase signature sequence present in the A subunit (from position 152 to position 183) is GGSSGGSAAAVAAGEVPF-SLGSDTGGSIRQPA (Fig.…”

“…6B). Amidases of this class have been studied from Rhodococcus, Brevibacterium, and Pseudomonas; these enzymes are important in the industrial conversion of certain amides into acids (26,27). Interestingly, glutamine or asparagine are not substrates for the Rhodococcus rhodochrous enzyme (27).…”

Enantioselective hydrolysis of racemic naproxen nitrile and naproxen amide to S-naproxen by new bacterial isolates

“…Highly enantioselective amidases have also been observed by other investigators with various a-substituted amides (Kieny L'Homme et al, 1981;Asano et al, 1989;Mayaux et al, 1990Mayaux et al, , 1991Kakeya et al, 1991;Cohen et al, 1992). In general, these activities were found after screening of strains from culture collections or in bacteria which were initially enriched with different often aliphatic nitriles or amides.…”

“…The enzymatic hydrolysis of nitriles represents a very convenient synthetic method for amides and/or carboxylic acids due to the mild reaction conditions. Furthermore, these enzymatic reactions also allow the enantioselective synthesis of optical active amides and carboxylic acids from racemic precursors (Mayaux et al, 1990(Mayaux et al, , 1991Yamamoto et al, 1990;Kakeya et al, 1991;Bianchi et al, 1991;Bhalla et al, 1992;Cohen et al, 1992;Layh et al, 1992).…”

Oligomerization of Sulfolobus solfataricus signature amidase is promoted by acidic pH and high temperature

“…The discovery of an amidase signature gene family (Mayaux et al 1991) allowed grouping of the primary structure of the codified enzymes into two classes, the first sharing a characteristic signature and the second lacking this signature. Amidase signature family enzymes include at least 200 proteins (most of these are putative, being derived from DNA data) from 90 different organisms distributed among Bacteria, Archaea and Eukarya (Altschul et al 1997).…”