Altered Metabolism of Polycyclic Aromatic Hydrocarbons by UDP-Glycosyltransferase 3A2 Missense Variants.

The UDP-glycosyltransferase (UGT) family of enzymes are important in the metabolism of a variety of exogenous substances including polycyclic aromatic hydrocarbons (PAHs), a potent class of environmental carcinogens. As compared to the majority of UGT enzymes, which utilize UDP-glucuronic acid as a cosubstrate, UGT3A2 utilizes alternative cosubstrates (UDP-glucose and UDP-xylose). UGT3A2 is expressed in aerodigestive tract tissues and was highly active against multiple PAHs with both cosubstrates. The goal of the present study was to assess the functional effects of UGT3A2 missense variants (MAF ≥ 0.005) on PAH metabolism and the utilization of cosubstrates. The glycosylation activity (Vmax/Km) of all variants against simple PAHs using both cosubstrates was significantly (P < 0.05) decreased by 42-100% when compared to wild-type UGT3A2. When utilizing UDP-glucose, the variant isoforms exhibited up to a 362-fold decrease in Vmax/Km when compared to wild-type UGT3A2, with a 3.1- to 14-fold decrease for D140N, A344T, and S435Y, a 24- and 43-fold decrease for A436T and R445C, respectively, and a 147- and 362-fold decrease for Y474C and Y74N, respectively. When utilizing UDP-xylose, the variants exhibited up to a 4.0-fold decrease in Vmax/Km when compared to wild-type UGT3A2; Y74N did not exhibit activity, and Y474C did not reach saturation (Km > 4000 µM). Additionally, both wild-type and variant UGT3A2 exhibited a significant (P < 0.05) difference in their utilization of UDP-glucose vs UDP-xylose as cosubstrates using 1-OH-pyrene as substrate. These data suggest that UGT3A2 missense variants decrease the detoxification of PAHs, potentially resulting in altered individual risk for PAH-related cancers.

Structural and Functional Characterization of Dynamic Oligomerization in Burkholderia cenocepacia HMG-CoA Reductase.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR), in most organisms, catalyzes the four-electron reduction of the thioester (S)-HMG-CoA to the primary alcohol (R)-mevalonate, utilizing NADPH as the hydride donor. In some organisms, including the opportunistic lung pathogen Burkholderia cenocepacia, it catalyzes the reverse reaction, utilizing NAD+ as a hydride acceptor in the oxidation of mevalonate. B. cenocepacia HMGR has been previously shown to exist as an ensemble of multiple non-additive oligomeric states, each with different levels of enzymatic activity, suggesting that the enzyme exhibits characteristics of the morpheein model of allostery. We have characterized a number of factors, including pH, substrate concentration, and enzyme concentration, that modulate the structural transitions that influence the interconversion among the multiple oligomers. We have also determined the crystal structure of B. cenocepacia HMGR in the hexameric state bound to coenzyme A and ADP. This hexameric assembly provides important clues about how the transition among oligomers might occur, and why B. cenocepacia HMGR, unique among characterized HMGRs, exhibits morpheein-like behavior.

Kinetic characterization of an oxidative, cooperative HMG-CoA reductase from Burkholderia cenocepacia.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is a key enzyme in endogenous cholesterol biosynthesis in mammals and isoprenoid biosynthesis via the mevalonate pathway in other eukaryotes, archaea and some eubacteria. In most organisms that express this enzyme, it catalyzes the NAD(P)H-dependent reduction of HMG-CoA to mevalonate. We have cloned and characterized the 6x-His-tagged HMGR from the opportunistic lung pathogen Burkholderia cenocepacia. Kinetic characterization shows that the enzyme prefers NAD(H) over NADP(H) as a cofactor, suggesting an oxidative physiological role for the enzyme. This hypothesis is supported by the fact that the Burkholderia cenocepacia genome lacks the genes for the downstream enzymes of the mevalonate pathway. The enzyme exhibits positive cooperativity toward the substrates of the reductive reaction, but the oxidative reaction exhibits unusual double-saturation kinetics, distinctive among characterized HMG-CoA reductases. The unusual kinetics may arise from the presence of multiple active oligomeric states, each with different Vmax values.

Surface histidine mutations for the metal affinity purification of a ß-carbonic anhydrase.

Metal affinity chromatography using polyhistidine tags is a standard laboratory technique for the general purification of proteins from cellular systems, but there have been no attempts to explore whether the surface character of a protein may be engineered to similar affinity. We present the Arg160His mutation of Haemophilus influenzae carbonic anhydrase (HICA), which mimics the endogenous metal affinity of Escherichia coli carbonic anhydrase (ECCA). The purity and activity of the mutant are reported, and the purification is discussed. This is the first step toward developing a general method to engineer surface metal affinity for use in purification and metal labeling techniques.