E-02E-02 — D2HGDH depletion: human oxidoreductase in EcN

Hypothesis

Human D-2-hydroxyglutarate dehydrogenase (D2HGDH), expressed in the colibactin-null EcN chassis with its mitochondrial targeting sequence removed, retains sufficient FAD-dependent dehydrogenase activity under micro-aerobic conditions (2–5% O₂) to deplete D-2-HG at concentrations present in vorasidenib-treated IDH-mutant glioma (0.37–2.6 mM).

Background

D2HGDH is the enzyme the human body uses to clear D-2-HG. It catalyzes the single-step oxidation: D-2-HG → α-ketoglutarate, using FAD as the electron acceptor. Loss-of-function mutations in D2HGDH cause D-2-hydroxyglutaric aciduria — confirming this is the primary human D-2-HG clearance mechanism.

Key advantages over the anaerobic dehydratase paths:

Property	E-02 (D2HGDH)	E-03 (LcdA/FldA)	E-04 (HgdABC)
Genes required	1	3	5
Substrate affinity (Km)	~0.1–0.5 mM	Unknown	~0.5–2 mM
O₂ requirement	Micro-aerobic	Strictly anaerobic	Strictly anaerobic
[4Fe-4S] cluster assembly	No	Yes	Yes

The estimated Km of ~0.1–0.5 mM (inferred from characterized family members; direct measurement is a primary readout of this experiment) makes D2HGDH the best candidate for the vorasidenib co-treatment scenario, where residual D-2-HG is 0.37–2.6 mM. A single gene with no iron-sulfur chemistry is also the simplest engineering path.

Kill criterion: If depletion activity at 5% O₂ drops below 10% of aerobic activity, D2HGDH is not viable as the primary depletion engine for this deployment context.

Depends on: E-01 (colibactin-null chassis must be complete before transformation).

Design

1. Clone D2HGDH — PCR-amplify human D2HGDH coding sequence minus the N-terminal mitochondrial targeting sequence (approximately first 29 amino acids). Clone into an EcN-compatible expression vector (pSC101 for low-copy stability; pUC origin for high-expression screening). Add C-terminal His₆ tag for purification and detection.
2. Transform into E-01 chassis — Electroporate into colibactin-null EcN. Confirm colony PCR for plasmid presence.
3. Confirm protein expression — SDS-PAGE and anti-His Western blot. Confirm band at expected molecular weight (~53 kDa for processed D2HGDH).
4. Purified enzyme assay — Affinity-purify His₆-D2HGDH. Measure dehydrogenase activity using FAD-dependent chemistry: primary readout = DCPIP reduction (600 nm, artificial electron acceptor) or FAD absorbance decrease at 450 nm. D2HGDH is FAD-dependent; NADH/340 nm assays are not applicable. Run across D-2-HG concentrations of 0.1, 0.5, 1, 2, 5, 10, 25, 50 mM. Fit Michaelis-Menten curve to determine Km and Vmax.
5. Whole-cell depletion assay — aerobic — Grow expressing strain to OD 0.6, wash, resuspend in minimal media supplemented with D-2-HG (0.5, 2, 5, 10 mM). Measure media D-2-HG by LC-MS/MS at 0, 4, 8, 24h.
6. Whole-cell depletion assay — micro-aerobic (5% O₂) — Repeat in anaerobic chamber at 5% O₂ / 5% CO₂ / 90% N₂.
7. Whole-cell depletion assay — low oxygen (1% O₂) — Repeat at 1% O₂.
8. Controls — Non-expressing EcN (empty vector) at each O₂ condition.

Estimated cost: $15,000–$25,000 USD. Timeline: Weeks 6–16 (dependent on E-01 completion).

Readouts

• D2HGDH protein expression confirmed by Western blot
• Km for D-2-HG from purified enzyme assay (target: ≤2 mM to match vorasidenib residual range)
• Vmax and estimated depletion rate under physiological conditions
• Whole-cell D-2-HG depletion rate at aerobic, 5% O₂, and 1% O₂
• Activity at 5% O₂ as percentage of aerobic (kill criterion: <10% = not viable)
• D-2-HG depletion to below 0.1 mM starting from vorasidenib-floor concentrations (0.5–2 mM)

Status notes

Planned. Depends on E-01. CRO must have certified anaerobic chamber for the low-oxygen conditions. Runs in parallel with E-03 (LcdA/FldA promiscuity) and E-04 (HgdABC pathway) — all three depletion paths are evaluated before Gate G1 enzyme selection at Week 14–16.