Recombinant Saccharomyces cerevisiae Porphobilinogen deaminase (HEM3)
Description
Introduction to Recombinant S. cerevisiae Porphobilinogen Deaminase (HEM3)
Porphobilinogen deaminase (EC 2.5.1.61), encoded by the HEM3 gene in S. cerevisiae, is the third enzyme in the heme biosynthesis pathway. Recombinant forms of this enzyme are produced through genetic modifications to enhance expression or stability. Its reaction mechanism involves the sequential condensation of four PBG molecules, releasing ammonia to form linear hydroxymethylbilane . This step is essential for synthesizing heme, a cofactor for oxygen transport and electron transfer proteins .
Reaction Equation
Metabolic Engineering Applications
Recombinant HEM3 has been engineered to address bottlenecks in heme biosynthesis. Key findings include:
Table 1: Heme Production in Engineered S. cerevisiae Strains
Overexpression of HEM3 alone triples heme output, but synergistic effects emerge when combined with genes like HEM13 (coproporphyrinogen III oxidase) or FET4 (iron transporter) .
Key Research Findings
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Genome-Scale Modeling: The ecYeast8 model identified HEM3 as a high-impact target, with flux balance analysis linking its activity to glycine and succinyl-CoA availability .
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CRISPR-Cas9 Engineering: A strain (IMX581-HEM3-HEM14-Δshm1) achieved a 70-fold heme increase by co-expressing HEM3 with other pathway genes .
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Cofactor Dependency: HEM3 requires a dipyrromethane cofactor for activity, which is auto-catalytically assembled from PBG . Mutations in this cofactor-binding domain (e.g., R167W) disrupt function and correlate with porphyria disorders .
Challenges and Limitations
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Regulatory Constraints: Native HEM3 expression is partially controlled by HAP2/3 transcription factors, requiring constitutive promoters (e.g., TEF1) for overexpression .
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Cofactor Stability: Insufficient heme or iron availability reduces enzyme efficiency, necessitating parallel engineering of Fe-S cluster biosynthesis .
Future Directions
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Protein Engineering: Rational design of HEM3 variants with improved catalytic efficiency or cofactor affinity .
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Systems Biology Approaches: Integration of multi-omics data to optimize heme flux in industrial strains .
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Biosensor Integration: Use of heme ligand-binding biosensors (Heme-LBB) for real-time monitoring of production strains .
Product Specs
Target Background
KEGG: sce:YDL205C
STRING: 4932.YDL205C
Q&A
Basic Research Questions
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What is the structure and function of porphobilinogen deaminase in Saccharomyces cerevisiae?
Porphobilinogen deaminase, encoded by the HEM3 gene in Saccharomyces cerevisiae, is the third enzyme in the heme biosynthetic pathway. It catalyzes the polymerization of four porphobilinogen molecules to form hydroxymethylbilane, a precursor in heme synthesis. The HEM3 gene contains an open reading frame of 981 nucleotides encoding a protein that shows extensive homology to porphobilinogen deaminase sequences from other organisms . This conservation indicates its evolutionary importance across species. The enzyme features a dipyrromethane cofactor that plays a crucial role in its catalytic mechanism, facilitating the step-wise polymerization of porphobilinogen molecules.
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How is the HEM3 gene regulated in Saccharomyces cerevisiae?
The regulation of HEM3 in S. cerevisiae involves several mechanisms. Contrary to earlier suggestions that HEM3 expression might be induced by porphobilinogen (its substrate), research has shown that its transcription is not affected by the cell's ability to produce porphobilinogen or heme . Instead, the HAP2 and HAP3 gene products play important roles in HEM3 expression regulation. A small region in the HEM3 promoter contains a sequence homologous to HAP2-3-4 binding sites found in several genes, including HEM1 . This suggests that HEM3 expression is regulated similarly to HEM1, which encodes the first enzyme in the heme biosynthetic pathway, indicating coordinated regulation of the pathway.
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What are the biochemical properties of purified porphobilinogen deaminase from S. cerevisiae?
Porphobilinogen deaminase from S. cerevisiae exhibits several distinct biochemical characteristics:
Property Value/Observation Molecular mass 30,000 ± 3,000 Da (with protease inhibitor) Alternative form 20,000 Da (less active, unstable, without protease inhibitor) Optimum pH 7.5-7.8 Kinetics Classical Michaelis-Menten Km for uroporphyrinogen 19 μM Vmax 3.6 nmol uroporphyrin/h Hill coefficient n = 1 The enzyme shows linear porphyrin formation with time and protein concentration . It is sensitive to heavy metals, which inhibit both porphyrin formation and porphobilinogen consumption. Interestingly, known sulfhydryl inactivating chemicals inhibit porphyrin formation without affecting porphobilinogen consumption. The enzyme is also completely inhibited by hydroxylamine, while ammonium ions have no effect on its activity .
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How does porphobilinogen deaminase compare between S. cerevisiae and other organisms?
Porphobilinogen deaminase shows varying degrees of sequence conservation across species. The S. cerevisiae HEM3 protein shares significant sequence identity with other organisms, though the level varies:
Organism Comparison Sequence Identity (%) S. cerevisiae vs. P. pastoris 54.0 Despite the conservation of the heme biosynthesis pathway, distinct differences exist among species. In E. coli, the formation of 5-aminolevulinic acid by ALA synthase (HEM1) is rate-limiting, whereas in S. cerevisiae, the HEM2 and HEM3-encoded enzymes (porphobilinogen synthase and deaminase) are considered rate-limiting . These differences influence strategies for enhancing recombinant heme protein production in different host organisms.
