Trapping of intermediates with substrate analog HBOCoA in the polymerizations catalyzed by class III polyhydroxybutyrate (PHB) synthase from Allochromatium vinosum

Abstract

Polyhydroxybutyrate (PHB) synthases (PhaCs) catalyze the formation of biodegradable PHB polymers that are considered as an ideal alternative to petroleum-based plastics. To provide strong evidence for the preferred mechanistic model involving covalent and noncovalent intermediates, a substrate analog HBOCoA was synthesized chemoenzymatically. Substitution of sulfur in the native substrate HBCoA with an oxygen in HBOCoA enabled detection of (HB)nOCoA (n = 2-6) intermediates when the polymerization was catalyzed by wild-type (wt-)PhaECAv at 5.84 h(-1). This extremely slow rate is due to thermodynamically unfavorable steps that involve the formation of enzyme-bound PHB species (thioesters) from corresponding CoA oxoesters. Synthesized standards (HB)nOCoA (n = 2-3) were found to undergo both reacylation and hydrolysis catalyzed by the synthase. Distribution of the hydrolysis products highlights the importance of the penultimate ester group as previously suggested. Importantly, the reaction between primed synthase [(3)H]-sT-PhaECAv and HBOCoA yielded [(3)H]-sTet-O-CoA at a rate constant faster than 17.4 s(-1), which represents the first example that a substrate analog undergoes PHB chain elongation at a rate close to that of the native substrate (65.0 s(-1)). Therefore, for the first time with a wt-synthase, strong evidence was obtained to support our favored PHB chain elongation model.

Figure 1

(A) Formation of (HB)_nCoA intermediates during incubation of 100 μM wt-PhaEC_Av with 20.0 mM HBOCoA at 30 °C for 1 (black), 2 (red), 3 (green), 4 (blue), 5 (orange), and 6 hrs (pink). HPLC condition is described in EXPERIMENTAL SECTION; (B) MALDI-TOF MS of species eluted at 9.80 (I), 29.0 (II), 37.7 (III), and 40.0–60.0 min (IV); (C) Assignment of peaks shown in B; (D) Conversion of the terminal HB unit into a crotonate in the presence of acid.

Figure 1

(A) Formation of (HB)_nCoA intermediates during incubation of 100 μM wt-PhaEC_Av with 20.0 mM HBOCoA at 30 °C for 1 (black), 2 (red), 3 (green), 4 (blue), 5 (orange), and 6 hrs (pink). HPLC condition is described in EXPERIMENTAL SECTION; (B) MALDI-TOF MS of species eluted at 9.80 (I), 29.0 (II), 37.7 (III), and 40.0–60.0 min (IV); (C) Assignment of peaks shown in B; (D) Conversion of the terminal HB unit into a crotonate in the presence of acid.

Figure 2

(A) Kinetic analysis of HBOCoA with 100 μM wt-PhaEC_Av. The data were fitted to Michaelis–Menten equation to give a K_M = 2.38 ± 0.23 mM and V_max = 9.73 ± 0.28 μM•min⁻¹. The reaction was performed in duplicate. (B) Rates of formation of (HB)₂OCoA (solid line) and (HB)₃OCoA (dash line) in the presence of 100 μM wt-PhaEC_Av and 20 mM HBOCoA. Linear regression of the data gave the slopes of 3.47 × 10⁻¹ and 3.51 × 10⁻² mM•hr-1 to (HB)₂OCoA and (HB)₃OCoA, respectively. The reaction was performed in duplicate.

Figure 3

(A) HPLC profiles of incubating 50.0 μM wt-PhaEC_Av with 5.00 mM (HB)₂OCoA (solid line) or (HB)₃OCoA (dash line) for 30 min. The t_R of CoAOH, HBOCoA, (HB)₂OCoA, and (HB)₃OCoA are 9.80, 17.7, 29.0, and 37.7 min, respectively; (B) Estimated rates of product formation in hr⁻¹; (C) Proposed pathways for product formation from incubation of (HB)_nOCoA (n = 2–3) with wt-PhaEC_Av.

Figure 4

(A) Typical HPLC (solid line) and radioactivity (bar) profiles of the reaction between 4.20 μM [³H]-sT-PhaEC_Av and 5.00 mM HBOCoA stopped by RQF. The dash line represents HPLC profile of synthesized standards, sT-O-CoA (44.1 min) and sTet-O-CoA (47.4 min); (B) MALDI-TOF MS of species eluted at 47.0-49.0 min. (C) Rate of formation of [³H]-sTet-O-CoA in the reaction between 4.20 μM [³H]-sT-PhaEC_Av and 5.00 mM HBOCoA. The data were fitted to y=a(1-e-^bx), where a = burst amplitude and b = k_obs. The fit gives an amplitude of 139.7±2.2 cpm (counts per minute) and a k_obs of 17.4±0.8 s⁻¹.

Scheme 1

PHA synthesis by PhaCs

Scheme 2

Mechanistic models of PHB chain elongation

Scheme 3

Strategies of using HBOCoA as a probe to test the preferred mechanistic model B

Scheme 4

Chemoenzymatic synthesis of HBOCoA and related analogs