Catalytic role of histidine-114 in the hydrolytic dehalogenation of chlorothalonil by Pseudomonas sp. CTN-3.

Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile; TPN) is an environmentally persistent fungicide that sees heavy use in the USA and is highly toxic to aquatic species and birds, as well as a probable human carcinogen. The chlorothalonil dehalogenase from Pseudomonas sp. CTN-3 (Chd, UniProtKB C9EBR5) degrades TPN to its less toxic 4-OH-TPN analog making it an exciting candidate for the development of a bioremediation process for TPN; however, little is currently known about its catalytic mechanism. Therefore, an active site residue histidine-114 (His114) which forms a hydrogen bond with the Zn(II)-bound water/hydroxide and has been suggested to be the active site acid/base, was substituted by an Ala residue. Surprisingly, ChdH114A exhibited catalytic activity with a kcat value of 1.07 s-1, ~ 5% of wild-type (WT) Chd, and a KM of 32 µM. Thus, His114 is catalytically important but not essential. The electronic and structural aspects of the WT Chd and ChdH114A active sites were examined using UV-Vis and EPR spectroscopy on the catalytically competent Co(II)-substituted enzyme as well as all-atomistic molecular dynamics (MD) simulations. Combination of these data suggest His114 can quickly and reversibly move nearly 2 Å between one conformation that facilitates catalysis and another that enables product egress and active site recharge. In light of experimental and computational data on ChdH114A, Asn216 appears to play a role in substrate binding and preorganization of the transition-state while Asp116 likely facilitates the deprotonation of the Zn(II)-bound water in the absence of His114. Based on these data, an updated proposed catalytic mechanism for Chd is presented.

Atrazine Degradation Using Immobilized Triazine Hydrolase from Arthrobacter aurescens TC1 in Mesoporous Silica Nanomaterials.

Triazine hydrolase fromArthrobacter aurescens TC1 (TrzN) was successfully immobilized on mesoporous silica nanomaterials (MSNs) for the first time. For both nonfunctionalized MSNs and MSNs functionalized with Zn(II), three pore sizes were evaluated for their ability to immobilize wild-type TrzN: Mobile composition of matter no. 41 (small, 3 nm pores), mesoporous silica nanoparticle material with 10 nm pore diameter (MSN-10) (medium, 6-12 nm pores), and pore-expanded MSN-10 (large, 15-30 nm pores). Of these six TrzN:MSN biomaterials, it was shown that TrzN:MSN-10 was the most active (3.8 ± 0.4 × 10-5 U/mg) toward the hydrolysis of a 50 µM atrazine solution at 25 °C. The TrzN:MSN-10 biomaterial was then coated in chitosan (TrzN:MSN-10:Chit) as chitosan has been shown to increase stability in extreme conditions such as low/high pH, heat shock, and the presence of organic solvents. TrzN:MSN-10:Chit was shown to be a superior TrzN biomaterial to TrzN:MSN-10 as it exhibited higher activity under all storage conditions, in the presence of 20% MeOH, at low and high pH values, and at elevated temperatures up to 80 °C. Finally, the TrzN:MSN-10:Chit biomaterial was shown to be fully active in river water, which establishes it as a functional biomaterial under actual field conditions. A combination of these data indicate that the TrzN:MSN-10:Chit biomaterial exhibited the best overall catalytic profile making it a promising biocatalyst for the bioremediation of atrazine.