Substrate Specificity and Engineering of Mevalonate 5-Phosphate Decarboxylase

Area of Science:

• Biochemistry
• Molecular Biology
• Microbial Metabolism

Background:

• The mevalonate (MVA) pathway is crucial for isoprenoid biosynthesis.
• A recently identified bifurcation in the MVA pathway involves an alternative route (altMVA) in Chloroflexi bacteria.
• This altMVA pathway utilizes a distinct decarboxylation step.

Purpose of the Study:

• To characterize the substrate specificity and structural basis of mevalonate 5-phosphate decarboxylase (MPD) from *Anaerolinea thermophila*.
• To compare MPD with mevalonate diphosphate decarboxylase (MDD) and delineate key residues for substrate specificity.
• To investigate the distribution of the altMVA pathway across different domains of life and engineer a synthetic biology platform.

Main Methods:

• Enzyme activity assays to determine catalytic efficiency for different substrates.
• X-ray crystallography to obtain near atomic-resolution structures of the enzyme bound to substrates.
• Extensive sequence and structural analyses to compare MPD and MDD enzymes.
• Metabolic engineering of *Saccharomyces cerevisiae*.

Main Results:

• The *A. thermophila* enzyme, annotated as MDD, exhibits dual specificity for both (R)-mevalonate 5-phosphate ((R)-MVAP) and (R)-mevalonate 5-diphosphate ((R)-MVAPP).
• X-ray crystal structures revealed the molecular basis for this dual substrate recognition.
• Key active-site residues were identified that distinguish MPD and MDD enzyme classes.
• Functional MPDs were found in halobacteria, previously misannotated as MDDs.
• No eukaryotic MPD candidates were identified, suggesting the altMVA pathway is absent in eukaryotes.

Conclusions:

• The study elucidates the functional and structural basis for substrate specificity in MPD and MDD enzymes.
• The alternative MVA pathway is present in certain bacteria and archaea but absent in eukaryotes.
• An engineered *Saccharomyces cerevisiae* strain utilizing the altMVA pathway serves as a valuable model and synthetic biology platform.