EL DERECHO A LA TUTELA JUDICIAL EFECTIVA EN LA LEGISLACIÓN COMPARADA:

Heterocyclisation of cysteine, serine and threonine proceeds via attack on the nucleotide triphosphate by a hemiorthoamide intermediate. However these complex structures reveal that the orientation of the nucleotide within the active site, relative to the incoming substrate is such that the ↵-phosphate is occluded from attack by the hemiorthoamide (Fig. 3.4), thus precluding an adenylation mechanism[51].

Chapter 3. Structure-guided Characterisation and Engineering of the Heterocyclase 75

Figure 3.4: Occlusion of ↵-phosphate The ↵-phosphate is occluded from attack of the reacting hemiorthoamide intermediate. (a) Incoming precursor peptide threads through to the active site, through the channel as shown, approaching the -phosphate. (b) Close-up view of the nucleotide in the active from the perspective of the incoming peptide. From this angle shielding of the↵-phosphate clearly precludes an adenylation mechanism. One monomer of LynD is shown as a purple cartoon. Domain 3 of the other monomer is represented with a pink surface. The precursor peptide is shown as a cyan cartoon. Nucleotide phosphates are shown as sticks coloured orange for phosphorous

and red for oxygen. Metal ions are shown as grey spheres.

To ensure that the observed orientation of the nucleotide is mechanistically relevant and to eliminate any possibility that it is an artefact of crystallisation, residues identified as nucleotide binding were mutated, and the mutants were tested for their ability to bind ATP or AMP and process substrate PatE0. Binding experiments were performed using ITC and can be seen in Fig. 3.5. The activity of each mutant was monitored using MALDI-TOF-MS. A summary of the binding and activity data can be seen in Table 3.1.

Chapter 3. Structure-guided Characterisation and Engineering of the Heterocyclase 76

Figure 3.5: Nucleotide bindingITC data for ATP and AMP titrated into LynD and LynD mutant solutions. The top panel shows raw data representing heat evolved in response to injections, the bottom panel shows the integrated heats of injections (⇤)

Chapter 3. Structure-guided Characterisation and Engineering of the Heterocyclase 77

Table 3.1: Nucleotide binding and processing of LynD mutants.

Nucleotide KD (µM)

Enzyme ATP AMP

No. of heterocycles (PatE0)

LynD 50.25 14.41 2

LynDK409A _{No binding No binding 2}

LynDK409E No binding No Binding 1

LynDE423R _{No binding 5.65 0}

LynDR427E No binding 23.04 1 and 2

LynDR636E _{No binding No binding 0, 1 and 2}

LynDR636A N/A No binding 0, 1 and 2

Both the K409A and K409E mutants completely abolished binding of ATP and AMP to LynD as determined by ITC experiments (Fig. 3.5, Table 3.1). Therefore the ability of each mutant to process PatE0substrate was unexpected (Table 3.1). The K409A mutant resulted in complete substrate turnover, converting both cysteine residues to thiazolines, while the K409E mutant slowed processing, catalysing only one cyclodehydration under standard conditions (Table 3.1). These data imply that both the K409A and K409E mutants do bind ATP, but only very weakly, below the detection limits of ITC, and that our standard reaction conditions contain sufficient ATP (5 mM) to facilitate catalysis. Consequently the ability of the K409A mutant to process substrate PatE0 was investigated again, using a lower ATP concentration (500µM), and the heterocyclisation reaction was monitored over time using MALDI-TOF-MS. By plotting the amount of product formed over time, as identified by MALDI-TOF-MS under these new reaction conditions, it becomes obvious that the K409A mutation impairs processing, forming both heterocycles more slowly compared with wild-type enzyme (Fig. 3.6).

The R636E and R636A mutations inhibit binding of both ATP and AMP to LynD (Fig. 3.5). Unfortunately the ITC experiment between LynDR636A _{and ATP reproducibly} resulted in the complex ITC curve as shown (Fig. 3.5), and could not be easily interpreted, and at present we are unable to explain this result. Both mutants significantly a↵ect substrate processing, resulting in a mixture of products containing

Chapter 3. Structure-guided Characterisation and Engineering of the Heterocyclase 78

Figure 3.6: LynDK409E _{heterocyclisation rate Relative rates of the}

heterocyclisation reaction between PatE0 and LynD + 5 mM ATP (blue), PatE0 and LynD + 500µM ATP (red) and PatE0 and LynDK409E_{+ 500µM (green) analysed by}

MALDI-TOF-MS. The top graph shows the time taken to form the 1st heterocycle and the bottom graphs shows the time taken to form the 2nd heterocycle. Each experiment was set up in triplicate and each measurement was repeated three times; thus each

time-point is an average of nine measurements. Errors are plotted as +/- 1 s.d..

0, 1 and 2 heterocycles. These data support the idea that R636 coordinates the ↵- phosphate, as observed in the crystal structure.

The E423R and the R427E mutations also impair substrate processing, forming 0 heterocycles, and a mixture of 1 and 2 heterocycles respectively, under standard reaction conditions. Interestingly, while both mutations completely abolish ATP binding, AMP

Chapter 3. Structure-guided Characterisation and Engineering of the Heterocyclase 79

still binds to each mutant (Fig. 3.5, Table 3.1). The E423R mutant binds AMP with higher affinity, and the R427E mutant shows only slightly weaker affinity than the wild- type enzyme (Table 3.1). The ability of the E423R and the R427E mutants to bind AMP, but not ATP, confirm the involvement of these residues in the coordination of and -phosphates as predicted by the crystal structure, indicating the observed orientation of the nucleotide in the active site is correct, and not an artefact of crystallisation. Consequently these data, strongly suggest heterocyclisation does not proceed via the previously proposed adenylation mechanism[51]_.