The thermophilic Bacillus smithii RAPc8 amidase can be potentially used for the production of a range of industrially useful acid products. The sterioselectivity of the enzyme enables the production of enantiopure acids that are difficult to produce by traditional chemical methods.
This project addresses two issues:
- To use structural analyses in order to develop an understanding of the molecular basis of the enzyme specificity.
- To use protein engineering methods to generate amidases with modified or improved specificity based on the structural information.
Amidases catalyse the hydrolysis of amides to the corresponding carboxylic acid and ammonia. They exist in all kingdoms of the living world but have been most extensively characterised amongst the bacteria.
A number of studies of amidase classification [cite]10.1016/S0167-4838(96)00145-8[/cite][cite]10.1046/j.1365-2672.2001.01378.x[/cite][cite]10.1186/gb-2001-2-1-reviews0001[/cite] have revealed that the bacterial aliphatic amidases (broadly classed as acylamide amidohydrolase, EC 22.214.171.124) are made up of two types.
The first group, the nitrilase-related family, includes the aliphatic amidases, hydrolysing only short-chain aliphatic amides [cite]10.1046/j.1365-2672.2001.01378.x[/cite]. The enzymes are typically homohexamers of approximately 230kDa, and contain a Cys166 residue (Pseudomonas aeruginosa amidase numbering), conserved across both nitrilase and amidase. This residue is believed to act as the catalytic nucleophile. The amidases from P. aeruginosa [cite]10.1016/0014-5793(87)80163-1[/cite][cite]10.1016/j.ijbiomac.2003.08.002[c/ite], Rhodococcus sp. R312 [cite source=pubmed]7958763[/cite], Bacillus stearothermophilus BR338 [cite]10.1016/S0141-0229(99)00150-7[/cite], Bacillus sp. BR449 [cite]10.1016/S0141-0229(00)00248-9[/cite] all belong to this group. Sequence comparison suggests that Bacillus smithii RAPc8 also belongs to the nitrilase superfamily (Figure 1, see additional attachments). We have also demonstrated using gel-exclusion chromatography that the amidase forms dimers (unpublished results). The catalytic triad residues (Cys-Glu-Lys), characteristic of the nitrilase superfamily, are conserved and structurally aligned between Bacillus smithii sp. RAPc8 and various members of the nitrilase superfamily (Figure1). However, the amidase differs from members of the nitrilase superfamily that are known to form spirals including nitrilase, cyanide dihydratase and cyanide hydratase by lacking two insertions (see figure1, supplementary material). These two insertions are thought to interact with the corresponding part of another subunit across a pseudo two-fold axis and thus lead to spiral formation and consequently activation of the nitrilase. It therefore seems unlikely that the amidase forms a spiral structure. Another key scientific question concerns the difference between the amidase and nitrilase active sites. The mechanisms involve the same three amino acids in a similar configuration. Only structure elucidation can provide the information necessary to begin answering this question.
Many amidases, including Rhodococcus sp. R312 wide spectrum amidase [cite]10.1271/bbb1961.50.2237[/cite] and Ps. aeruginosa wide spectrum amidase [cite]10.1016/S0065-2911(08)60442-7[/cite] have the ability not only to transfer the acyl moiety of the amide to water, but also to hydroxylamine to form hydroxamates. Not only does the amide transfer reaction have significant industrial potential [cite]10.1046/j.1365-2672.2001.01378.x[/cite], but has greatly facilitated the study of amidase reaction mechanisms. It was demonstrated that amides react with the enzyme to give an acyl-enzyme complex, which is then subject to nucleophilic attack by the hydroxylamine co-substrate [cite]10.1016/S0065-2911(08)60442-7[/cite][cite]10.1271/bbb1961.50.2237[/cite]. It is therefore assumed that the mechanism for amide hydrolysis should be the same. Once the acyl-enzyme intermediate is formed, nucleophilic attack leads to the transfer of the acyl group to the co-substrate (water or hydroxylamine), leading to the formation of carboxylates or hydroxymates. Our proposed amidase reaction mechanism is shown in figure 2 (see additional attachments). Amide preparation is essentially the reverse of hydrolysis, and a crucial factor is the nature of the amine-nucleophile reactant which must effect amino-substitution in an acyl or carboxylic acid substrate.
No three-dimensional structures have been reported for the amidases and the structural basis for its catalytic activity and specificity remains unclear.