Structural insights into Xanthomonas campestris pv. campestris NAD+ biosynthesis via the NAM salvage pathway.

Nicotinamide phosphoribosyltransferase (NAMPT) plays an important role in the biosynthesis of nicotinamide adenine dinucleotide (NAD+) via the nicotinamide (NAM) salvage pathway. While the structural biochemistry of eukaryote NAMPT has been well studied, the catalysis mechanism of prokaryote NAMPT at the molecular level remains largely unclear. Here, we demonstrated the NAMPT-mediated salvage pathway is functional in the Gram-negative phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc) for the synthesis of NAD+, and the enzyme activity of NAMPT in this bacterium is significantly higher than that of human NAMPT in vitro. Our structural analyses of Xcc NAMPT, both in isolation and in complex with either the substrate NAM or the product nicotinamide mononucleotide (NMN), uncovered significant details of substrate recognition. Specifically, we revealed the presence of a NAM binding tunnel that connects the active site, and this tunnel is essential for both catalysis and inhibitor binding. We further demonstrated that NAM binding in the tunnel has a positive cooperative effect with NAM binding in the catalytic site. Additionally, we discovered that phosphorylation of the His residue at position 229 enhances the substrate binding affinity of Xcc NAMPT and is important for its catalytic activity. This work reveals the importance of NAMPT in bacterial NAD+ synthesis and provides insights into the substrate recognition and the catalytic mechanism of bacterial type II phosphoribosyltransferases.

Characterization of a novel hyper-thermostable and chlorpyrifos-hydrolyzing carboxylesterase EstC: A representative of the new esterase family XIX.

Carboxylesterases have widely been used in a series of industrial applications, especially, the detoxification of pesticide residues. In the present study, EstC, a novel carboxylesterase from Streptomyces lividans TK24, was successfully heterogeneously expressed, purified and characterized. Phylogenetic analysis showed that EstC can be assigned as the first member of a novel family XIX. Multiple sequence alignment indicated that EstC has highly conserved structural features, including a catalytic triad formed by Ser155, Asp248 and His278, as well as a canonical Gly-His-Ser-Ala-Gly pentapeptide. Biochemical characterization indicated that EstC exhibited maximal activity at pH 9.0 (Tris-HCl buffer) and 55 °C. It also showed higher activity towards short-chain substrates, with the highest activity for p-nitrophenyl acetate (pNPA2) (Km = 0.31 ± 0.02 mM, kcat/Km = 1923.35 ± 9.62 s-1 mM-1) compared to other pNP esters used in this experiment. Notably, EstC showed hyper-thermostability and good alkali stability. The activity of EstC had no significant changes when it was incubated under 55 °C for 100 h and reached half-life after incubation at 100 °C for 8 h. Beyond that, EstC also showed stability at pH ranging from 6.0 to 11.0 and about 90% residual activity still reserved after treatment at pH 8.0 or 9.0 for 26 h, especially. Furthermore, EstC had outstanding potential for bioremediation of chlorpyrifos-contaminated environment. The recombinant enzyme (0.5 U mL-1) could hydrolyze 79.89% chlorpyrifos (5 mg L-1) at 37 °C within 80 min. These properties will make EstC have a potential application value in various industrial productions and detoxification of chlorpyrifos residues.

Optical performance of toric intraocular lenses in the presence of decentration.

AIM: To evaluate the optical performance of toric intraocular lenses (IOLs) after decentration and with different pupil diameters, but with the IOL astigmatic axis aligned. METHODS: Optical performances of toric T5 and SN60AT spherical IOLs after decentration were tested on a theoretical pseudophakic model eye based on the Hwey-Lan Liou schematic eye using the Zemax ray-tracing program. Changes in optical performance were analyzed in model eyes with 3-mm, 4-mm, and 5-mm pupil diameters and decentered from 0.25 mm to 0.75 mm with an interval of 5° at the meridian direction from 0° to 90°. The ratio of the modulation transfer function (MTF) between a decentered and a centered IOL (MTFDecentration/MTFCentration) was calculated to analyze the decrease in optical performance. RESULTS: Optical performance of the toric IOL remained unchanged when IOLs were decentered in any meridian direction. The MTFs of the two IOLs decreased, whereas optical performance remained equivalent after decentration. The MTFDecentration/MTFCentration ratios of the IOLs at a decentration from 0.25 mm to 0.75 mm were comparable in the toric and SN60AT IOLs. After decentration, MTF decreased further, with the MTF of the toric IOL being slightly lower than that of the SN60AT IOL. Imaging qualities of the two IOLs decreased when the pupil diameter and the degree of decentration increased, but the decrease was similar in the toric and spherical IOLs. CONCLUSIONS: Toric IOLs were comparable to spherical IOLs in terms of tolerance to decentration at the correct axial position.