Redesign of BVMOs

For enzyme redesign studies, structural information of a targeted biocatalyst is very valuable. However, at present, no structure of the well-studied BVMO, CHMO, is available. In order to obtain a predictive structural model of the active site of CHMO, a cubic space model has been built based on the substrate acceptance of this biocatalyst.53 While this model can predict to some extent the specificity and selectivity of CHMO, it is of little use for enzyme redesign studies. However, even if no structural information is available, it is still possible to exploit random mutagenesis methods in order to change specific biocatalytic properties of an enzyme. Such an approach of directed evolution has been performed with CHMO by the Reetz group.54 By error-prone PCR, a library of CHMO mutants was created. By using chiral GC analysis, a library of 10 000 mutants was screened for improved enantioselectivity using the prochiral substrate 4-hydroxycyclohexanone. Upon Baeyer-Villiger oxidation by wild-type CHMO, this ketone is converted into both corresponding lactone enantiomers (only 9% ee for the (R)-enantiomer). The same library was also used to find mutants that show an improved enantioselectivity with methyl-p-methylbenzyl thioether. This sulfide is readily oxidized by wildtype CHMO forming the (R)-sulfoxide in low enantiomeric excess (ee = 14%). Although only a relatively small library of mutants was screened due to the limited screening efficiency (800 mutants per day), impressive results were obtained. For both reactions, multiple improved mutants were found that could be improved even further in a second round of mutagenesis. For the Baeyer-Villiger reaction, the enantioselectivity could be improved from 9 to 90% ee while mutants with the opposite enantioselectivity could also be retrieved. For the sulfoxidation reaction, several mutants were found that showed an excellent enantioselective behavior (>98% ee). Interestingly, both screens yielded mutants with improved biocatalytic properties in which only one specific residue was replaced: F432. Most other retrieved mutants contained multiple mutations suggesting that multiple mutations (additive effects) are needed to improve enantioselectivity.

Except for the directed evolution study on CHMO, only a few other enzyme redesign studies on BVMOs have been reported. By sequence alignment of BVMO sequences, residues have been identified as targets to change the coenzyme specificity of CHMO and HAPMO.55 As mentioned above, most BVMOs are only active with the relatively expensive coenzyme NADPH, thereby compromising cost-effective biocatalytic applications. It would be advantageous to engineer BVMOs which are active with NADH. By site-directed mutagenesis, it was found that one specific residue (K326 in CHMO) is crucially involved in recognizing the 2 -phosphate of NADPH. This lysine residue is conserved in all BVMO sequences which is in line with the observation that all type I BVMOs are NADPH-specific. However, while it was possible to change the selectivity of both CHMO and HAPMO toward NADH, all prepared mutants showed a modest to low activity with NADH. This indicates that a more thorough enzyme redesign strategy is needed in which the presently available structural information could be exploited (see below). Another site-directed mutagenesis study was performed by Cheesman et al.56 in which all histidine residues in CHMO were replaced to probe their respective function. This revealed that replacement of H59 prevented expression while the H163Q mutant exhibited only 10% activity. This latter observation is in line with the fact that H163 is part of the BVMO-specific motif mentioned above. Replacing the corresponding histidine in HAPMO also affected the activity of the respective enzyme strongly.39

Other sequence-function relationship data concerning a type I BVMO come from studies that focused on elucidating the genetic basis of drug resistance of M. tuberculosis isolates toward thioamides.45 46 It was found that a range of point mutations in a specific gene, etaA, were associated with thioamide drug resistance. The resulting drug resistance can be ascribed to the inactivation of the corresponding enzyme: ethionamide monooxygenase. Future studies will reveal whether the reported mutations are fatal for expression or result in inactive enzyme. Ethionamide monooxygenase represents a type I BVMO and is able to convert a range of ketones into the corresponding esters.47 Except for catalyzing Baeyer-Villiger oxidations, the enzyme is also able to oxidize the sulfide moieties of several antitubercular thioamide drugs. The oxidized drugs appear to be highly toxic for mycobacteria. This indicates that ethionamide monooxygenase acts as a prodrug activator. In vitro, ethionamide monooxygenase only displays a very low activity with all tested substrates. This low activity may be due to the fact that all tested substrates are unrelated to the (unknown) physiological substrate of ethionamide monooxygenase. However, it might also indicate that ethionamide monooxygenase needs other components to be fully active.

As mentioned above, the first BVMOs were already purified several decades ago. Subsequent biochemical studies have revealed that these enzymes are typically soluble and often easy to express at high levels in, e.g., E. coli. These features suggest that BVMOs are perfect candidates for X-ray crystallography studies. However, crystallization of several type I BVMOs has been attempted and proven to be difficult. Several groups have tried to crystallize the prototype BVMO: CHMO from Acinetobacter. However, all attempts have been without success, which is probably caused by the relative instability of this specific monooxy-genase. This is in line with the observation that CHMO is sensitive to cysteine oxidation leading to enzyme inactivation.57 Also, ethionamide monooxygenase has been subjected to crystallization trials. Unfortunately, the recombinant enzyme withstands crystallization as it tends to aggregate in its pure form (M. W. Fraaije, personal communication). We have successfully crystallized HAPMO resulting in large well-shaped yellow crystals. However, X-ray analysis of these crystals revealed that the diffracting power was very poor with a resolution limit of 6 A on a synchrotron source. As flexibility in N-termini has been shown to introduce heterogeneity in protein crystals, truncation could be a solution for the diffraction problem as HAPMO contains an extended N-terminus when compared with other BVMOs.58 To test this, several truncated HAPMO mutants were also crystallized.52 However, no improvement in diffraction properties was found.

Several BVMOs have been examined with respect to their stability. This has revealed that CHMO is not a very stable biocatalyst. The enzyme is rapidly degraded intracellularly when expressed in E. coli.59 In its isolated form, the enzyme is also quite unstable: t^ = 24h at 25°C.60 Additives, e.g., kosmotropic salts, have a stabilizing effect but are not desirable for biocatalytic applications. It was also reported that immobilization on a solid carrier is a more effective way to stabilize CHMO. HAPMO was shown to inactivate rapidly at elevated temperatures: t\fl = 80 min at 36°C.16 For this BVMO, it was shown that the lifetime could be increased 4-fold by adding the FAD cofactor. Addition of a NADPH coenzyme analog resulted in a more drastic effect as the enzyme remained fully active for 120 min at 36°C. While the addition of a coenzyme analog will inhibit efficient catalysis, it can be used as additive when a BVMO has to be stored. In order to obtain a more robust BVMO, we decided to search for a BVMO gene from a thermophilic microorganism. While no BVMO genes could be identified in hyperthermophilic archaea (Table 2), we discovered two genes in the genome of the semi-thermophile T. fusca (see above). One of the identified BVMOs was found to be overexpressed in E. coli as a soluble, fully flavinylated and active enzyme.17 As indicated above, the enzyme was found to be highly active on phenylacetone and is thermostable. Only at temperatures above 50°C, it tends to inactivate (ti/ = 24 h at 52°C). Possibly due to its robustness, PAMO readily crystallizes yielding crystals with good diffraction properties (<2A). This resulted in the elucidation of its crystal structure in 2004.61 The availability of the PAMO structure offers new possibilities for redesigning this or other BVMOs. One structure-inspired enzyme redesign study concerning PAMO has already been reported.62 By sequence alignment of CHMO and PAMO, it was deduced that PAMO contains an extended active site loop when compared with CHMO. It was probed whether, by deleting one or two loop residues, the substrate acceptance of PAMO could be altered. The designed mutants indeed brought about significant changes in substrate acceptance.

While wild-type PAMO was unable to convert 2-phenylcyclohexanone efficiently, all deletion mutants readily accepted this ketone as substrate. All mutants also displayed a similar thermostability when compared with the parent enzyme. The most active mutant (deletion of S441 and A442) was used for examining its enantioselective properties. It was found that the mutant preferably formed the (R)-enantiomer of the corresponding lactone (E = 100). While CHMO also shows a similar enantioselective behavior, this PAMO deletion mutant is a better candidate for future applications due to its superior stability. This clearly demonstrates that PAMO can be used as parent enzyme to design thermostable BVMO variants. It also illustrates that the available crystal structure of PAMO will be of great help for BVMO redesign efforts.5

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