Implementación de Diagnostic exercise

Hans Krebs noted that D-amino acids could be rapidly deaminated when incubated with fresh slices of rat kidney and liver, while naturally occurring l-isomers were also catalysed. He showed that the factor involved in action on non-naturally occurring amino acids could be extracted from fresh or dry tissue (while the enzyme acting on l-isomers was inhibited by the purification steps). This was the D-amino acid oxidase (Krebs, 1935). A prototype of the oxidase class of flavoproteins, DAAO was found to catalyse the oxidative deamination of non-acidic D-amino acids to their corresponding α-ketoacids. The most preferred substrates of this enzyme include amino acids with small hydrophobic side chains, followed by those bearing polar, aromatic and basic groups (Pollegioni et al., 1992). As a stable homodimer with tightly bound flavin adenine nucleotide (FAD) molecule, this 40kDa protein, can be used as a means to analyse D-amino acids in various body regions. In the brain this enzyme becomes more selective due to the narrow expression of D-amino acids here. To date only one amino acid is shown to be present in significant amounts in brain tissue, upon which the DAAO acts: D-serine. Thus this enzyme can be used to make a biosensor which will selectivity detect D-serine.

DAAO from a number of sources has since been discovered including humans, porcine kidney, Trigonopsis variabilis, Rhodotorula gracilis, Candida boidinii and

Fusarium solani. These enzymes differ in stability, substrate preference and specificity as well as in binding site kinetics. Unfortunately, direct comparisons of the kinetic parameters among these DAAOs is not feasible since published data has

55 been collected using different techniques and under different experimental conditions. However, only the mammalian source of the enzyme is commercially available, and easy methods for expression and purification of other DAAOs have only in the last 5-10years become available. Some of the earliest protocols for example, for DAAORg purification lasted 4-5 days, with native expression of the protein in yeast, followed by separation with ammonium sulphate, DEAE-sepharose and Mono S columns. As seen with native protein purifications, the yield was poor (Pilone Simonetta et al., 1989a; Pollegioni & Pilone, 1992). This purification step was greatly improved by the purification of cDNA from the yeast, which was inserted into a plasmid to be expressed in E. coli. The protein was found to catalytically active and soluble, with much greater yield of protein (Pollegioni et al., 1997). But the greatest advancement came in the form a histidine tag, which could be encoded onto the protein C-terminal, allowing a single-step purification using a nickel column (Molla et al., 1998).

DAAORg has a number of unique qualities, including highly efficient catalysis and tight binding with the coenzyme FAD, that make it more efficient at oxidising D - amino acids (and D-serine) compared to other sources of the enzyme (Pilone Simonetta et al., 1989b; Pollegioni et al., 2002). Unsurprisingly, the turnover numbers (with D-Ala as substrate) determined are highest for DAAORg 345 s-1, compared to DAAOTv and DAAOPk, 52.5 and 12.7 s-1 respectively (Porter et al., 1977; Pollegioni et al., 1992; Tishkov & Khoronenkova, 2005). Furthermore, it is more stable in an immobilised form and can best withstand changes in temperature and pH when compared to enzyme from T. variabilis and porcine kidney (Pilone Simonetta et al., 1989b; Pollegioni et al., 2002; Pollegioni et al., 2004). It is believed that the specific presence of a 23-residue C-terminal loop (βF5-βF6), is responsible for correct dimeric formation, that is accounts for higher stability of DAAORg (Pollegioni et al., 2002). A further peculiar feature of the DAAORg structure is the absence of a loop acting as an active site ‘lid’. In fact, in the mammalian enzyme, the conformational change of this ‘lid’ ( loop βI5-βI6) allows the substrate/product exchange at the active site: Indeed, the dissociation of the product from the enzyme is the rate-limiting step in catalysis (Porter et al., 1977). In DAAORg, the active site entrance is only partially hindered by the flexible side chain of a tyrosine (Tyr238), resulting in a faster exchange and a more efficient enzyme (see above).

56 The commercially available DAAOPk was widely used in assays and for fabrications of biosensors, but it showed low stability, and a low turnover number. D-serine biosensors formed from this enzyme were not sensitive enough for used in the brain due to the low detection limits, in the 100µM range; slow response and poor stability (Johansson et al., 1993; Jianzhong et al., 1994). But a surprising paper published recently has utilised this enzyme to make a sensor that is by far the most sophisticated biosensor made using DAAOpk. Sensitivity was reported to be 61± 7µA mM-1 cm-2, LOD of 20nM and very fast response (Zain et al., 2010), suggesting that although the enzyme is a contributing factor to the features of a biosensor, other dynamics are also involved. But a biosensor made using the same cross-linking method and DAAORg enzyme shows enhanced features, including a LOD of 16µM (theoretical) and sensitivity of 89± 33µA mM-1 cm-2 (Pernot et al., 2008).

Conclusively, then, a limiting factor in making a D-serine biosensor sensitive enough for use in the brain has been the availability of a more stable and active form of DAAO. Although, even now, no other source of DAAO is commercially available, the advances in expression and purification technology make other sources of DAAOs more accessible, leading to better D-serine biosensors.