Delta-Aminolevulinic Acid Dehydratase Enzyme Activity and Susceptibility to Lead Toxicity in Uganda’s Urban Children

Rapid industrialization, urbanization, and population explosion in sub-Saharan Africa escalate environmental Lead levels with subsequent elevation of blood Lead levels in children. Nutrition status, age, and genetics govern one’s susceptibility to Lead toxicity. This study expounded this susceptibility by relating blood Lead levels, d-aminolevulinic acid dehydratase enzyme activity (ALAD), and genetic variations of proteins that code for ALAD enzyme in urban children of Uganda. Spectrophotometric analysis for blood Lead (BL), hemoglobin levels, and determination d-levels aminolevulinic acid dehydratase enzyme activity of the blood samples from 198 children were performed prior to a polymerase chain reaction and restriction fragment length digestion for ALAD polymorphism was done. Up to 99.5% of samples coded for the ALAD1 allele whereas 0.05% coded for ALAD2. Genotypes ALAD2-2 members had elevated BLL (mean 14.1 µg/dL) and reduced ALAD enzyme activity compared to others. This, therefore, implies that the majority of children hoard BL which may affect them later in life.

Enhanced In Vitro Cascade Catalysis of Glycerol into Pyruvate and Acetoin by Integration with Dihydroxy Acid Dehydratase from Paralcaligenes ureilyticus

Recently, an in vitro enzymatic cascade was constructed to transform glycerol into the high-value platform chemical pyruvate. However, the low activity of dihydroxy acid dehydratase from Sulfolobus solfataricus (SsDHAD) limited the efficiency. In this study, the enzymatic reduction of pyruvate catalyzed by d-lactate dehydrogenase from Pseudomonas aeruginosa PAO1 was used to assay the activities of dihydroxy acid dehydratases. Dihydroxy acid dehydratase from Paralcaligenes ureilyticus (PuDHT) was identified as the most efficient candidate for glycerate dehydration. After the optimization of the catalytic temperature for the enzymatic cascade, comprising alditol oxidase from Streptomyces coelicolor A3, PuDHT, and catalase from Aspergillus niger, 20.50 ± 0.27 mM of glycerol was consumed in 4 h to produce 18.95 ± 0.97 mM of pyruvate with a productivity 12.15-fold higher than the previous report using SsDHAD. The enzymatic cascade was further coupled with the pyruvate decarboxylase from Zymomonas mobile for the production of another platform compound, acetoin. Acetoin at a concentration of 8.52 ± 0.12 mM was produced from 21.62 ± 0.19 mM of glycerol with a productivity of 1.42 ± 0.02 mM h−1.

Engineering of a thermophilic dihydroxy-acid dehydratase to enhance its dehydration ability on glycerate to pyruvate and its application in in vitro synthetic enzymatic biosystems

The low activity of dihydroxy-acid dehydratase (DHAD) on dehydration of glycerate to pyruvate hampers its applications in the biosystems. Protein engineering of a thermophilic DHAD from Sulfolobus solfataricus (SsDHAD) was performed to increase its dehydratation activity. A novel high-throughput method was established. A triple-mutant (I161M/Y145S/G205K) with a 10-fold higher activity on glycerate dehydration was obtained after three rounds of iterative saturation mutagenesis (ISM) based on computational analysis. The shrunk substrate-binding pocket and newly formed hydrogen bonds were the reason for the activity improvement of the mutant. For the in vitro synthetic enzymatic biosystems of converting glucose or glycerol to L-lactate, the biosystems with the mutant SsDHAD showed 3.32- and 2.34-times of the reaction rate than that of wild type, respectively. This study demonstrates the potential of protein engineering to improve the efficiency of in vitro synthetic enzymatic biosystems by enhancing the enzyme activity of rate-limited enzymes.

Heme biosynthesis depends on previously unrecognized acquisition of iron-sulfur cofactors in human amino-levulinic acid dehydratase

Heme biosynthesis and iron-sulfur cluster (ISC) biogenesis are two major mammalian metabolic pathways that require iron. It has long been known that these two pathways interconnect, but the previously described interactions do not fully explain why heme biosynthesis depends on intact ISC biogenesis. Herein we identify a previously unrecognized connection between these two pathways through our discovery that human aminolevulinic acid dehydratase (ALAD), which catalyzes the second step of heme biosynthesis, is an Fe-S protein. We find that several highly conserved cysteines and an Ala306-Phe307-Arg308 motif of human ALAD are important for [Fe4S4] cluster acquisition and coordination. The enzymatic activity of human ALAD is greatly reduced upon loss of its Fe-S cluster, which results in reduced heme biosynthesis in human cells. As ALAD provides an early Fe-S-dependent checkpoint in the heme biosynthetic pathway, our findings help explain why heme biosynthesis depends on intact ISC biogenesis.

Use of Generalized Additive Model to Detect the Threshold of δ-Aminolevulinic Acid Dehydratase Activity Reduced by Lead Exposure

Background: Lead inhibits the enzymes in heme biosynthesis, mainly reducing δ-aminolevulinic acid dehydratase (ALAD) activity, which could be an available biomarker. The aim of this study was to detect the threshold of δ-aminolevulinic acid dehydratase activity reduced by lead exposure. Methods: We collected data on 121 lead workers and 117 non-exposed workers when annual health examinations were performed. ALAD activity was determined by the standardized method of the European Community. ALAD G177C (rs1800435) genotyping was conducted using the polymerase chain reaction and restricted fragment length polymorphism (PCR-RFLP) method. In order to find a threshold effect, we used generalized additive models (GAMs) and scatter plots with smoothing curves, in addition to multiple regression methods. Results: There were 229 ALAD1-1 homozygotes and 9 ALAD1-2 heterozygotes identified, and no ALAD2-2 homozygotes. Lead workers had significantly lower ALAD activity than non-exposed workers (41.6 ± 22.1 vs. 63.3 ± 14.0 U/L, p < 0.001). The results of multiple regressions showed that the blood lead level (BLL) was an important factor inversely associated with ALAD activity. The possible threshold of BLL affecting ALAD activity was around 5 μg/dL. Conclusions: ALAD activity was inhibited by blood lead at a possible threshold of 5 μg/dL, which suggests that ALAD activity could be used as an indicator for lead exposure regulation.