l-Amino acid oxidase as biocatalyst: a dream too far?

Abstract

L-amino acid oxidase (LAAO) is a flavoenzyme containing non-covalently bound flavin adenine dinucleotide, which catalyzes the stereospecific oxidative deamination of l-amino acids to α-keto acids and also produces ammonia and hydrogen peroxide via an imino acid intermediate. LAAOs purified from snake venoms are the best-studied members of this family of enzymes, although a number of LAAOs from bacterial and fungal sources have been also reported. From a biochemical point of view, LAAOs from different sources are distinguished by molecular mass, substrate specificity, post-translational modifications and regulation. In analogy to the well-known biotechnological applications of d-amino acid oxidase, important results are expected from the availability of suitable LAAOs; however, these expectations have not been fulfilled yet because none of the "true" LAAOs has successfully been expressed as a recombinant protein in prokaryotic hosts, such as Escherichia coli. In enzyme biotechnology, recombinant production of a protein is mandatory both for the production of large amounts of the catalyst and to improve its biochemical properties by protein engineering. As an alternative, flavoenzymes active on specific l-amino acids have been identified, e.g., l-aspartate oxidase, l-lysine oxidase, l-phenylalanine oxidase, etc. According to presently available information, amino acid oxidases with "narrow" or "strict" substrate specificity represent as good candidates to obtain an enzyme more suitable for biotechnological applications by enlarging their substrate specificity by means of protein engineering.

Structure-Function Relationships in l-Amino Acid Deaminase, a Flavoprotein Belonging to a Novel Class of Biotechnologically Relevant Enzymes

“…The far-UV circular dichroism spectrum shows that PmaLAAD-00N is folded and contains secondary structure elements (data not shown). Amino acid oxidases are known to interact with a number of carboxylic acids, yielding specific spectral modifications and thus representing useful active-site probes (4,31,32). The binding to PmaLAAD-00N of various compounds known as DAAO and/or LAAO active-site ligands (14,33,34) was investigated in the absence and in the presence of E. coli membranes (corresponding to Ϸ0.30 mg of cells for each microgram of Pma-LAAD-00N).…”

“…These flavoenzymes catalyze an irreversible reaction (differently from aminotransferases) and do not require a specific step of cofactor regeneration, as otherwise required by the NAD-dependent dehydrogenases. However, because of the problems associated with overexpression of snake venom LAAOs in recombinant hosts and the limited substrate acceptance of the microbial counterparts, no appropriate LAAOs for biocatalysis are available (4).…”

l-Hydroxyproline andd-Proline Catabolism in Sinorhizobium meliloti

“…predicted to be a cis-4-D-Hyp oxidase/dehydrogenase (14), and several proteins are annotated as D-amino acid oxidase enzymes in S. meliloti, e.g., Smb20877 and Smc03265. As these enzymes generally have broad specificity (51), it seems likely that the residual growth of the hypO mutant on trans-4-L-Hyp is due to these oxidase activities; however, those enzymes were not examined further. A cis-4-D-Hyp dehydrogenase enzyme from P. putida was recently purified and characterized (12), and the sequence of that protein (PP1255) is 35% identical to that of the S. meliloti HypO protein.…”

L-Amino Acid Oxidase, a Novel Antibacterial Protein in the Rockfish <i>Sebastes schlegeli</i>

“…LAOs are widely distributed across diverse phyla from bacteria to mammals (Hughes 2010;Pollegioni et al 2013). LAOs in microorganisms appear to be involved in the utilization of ammonia as a nitrogen source, and those in animals show distinct biologic and physiologic effects such as antimicrobial activity, antiparasitic activity, apoptosis, cytotoxicity, edema, hemolysis, hemorrhage and platelet aggregation (Du and Clemeston 2002).…”