Analysis of the polymerization initiation and activity of Pasteurella multocida heparosan synthase PmHS2, an enzyme with glycosyltransferase and UDP-sugar hydrolase activity

Abstract

Heparosan synthase catalyzes the polymerization of heparosan (-4GlcUAβ1-4GlcNAcα1-)(n) by transferring alternatively the monosaccharide units from UDP-GlcUA and UDP-GlcNAc to an acceptor molecule. Details on the heparosan chain initiation by Pasteurella multocida heparosan synthase PmHS2 and its influence on the polymerization process have not been reported yet. By site-directed mutagenesis of PmHS2, the single action transferases PmHS2-GlcUA(+) and PmHS2-GlcNAc(+) were obtained. When incubated together in the standard polymerization conditions, the PmHS2-GlcUA(+)/PmHS2-GlcNAc(+) showed comparable polymerization properties as determined for PmHS2. We investigated the first step occurring in heparosan chain initiation by the use of the single action transferases and by studying the PmHS2 polymerization process in the presence of heparosan templates and various UDP-sugar concentrations. We observed that PmHS2 favored the initiation of the heparosan chains when incubated in the presence of an excess of UDP-GlcNAc. It resulted in a higher number of heparosan chains with a lower average molecular weight or in the synthesis of two distinct groups of heparosan chain length, in the absence or in the presence of heparosan templates, respectively. These data suggest that PmHS2 transfers GlcUA from UDP-GlcUA moiety to a UDP-GlcNAc acceptor molecule to initiate the heparosan polymerization; as a consequence, not only the UDP-sugar concentration but also the amount of each UDP-sugar is influencing the PmHS2 polymerization process. In addition, it was shown that PmHS2 hydrolyzes the UDP-sugars, UDP-GlcUA being more degraded than UDP-GlcNAc. However, PmHS2 incubated in the presence of both UDP-sugars favors the synthesis of heparosan polymers over the hydrolysis of UDP-sugars.

FIGURE 1.

Influence of the UDP-sugar concentration on the polymerization process of the combined PmHS2 single action transferases. PmHS2-GlcUA⁺/PmHS2-GlcNAc⁺ (1:1) (45 μg/ml each) were incubated together for 24 h in the presence of 1, 5, and 20 mm concentrations of each UDP-sugar, respectively. The chain length and size distribution of heparosan polymer is assessed with a Novex 20% TBE gel.

FIGURE 2.

Influence of the incubation temperature on the polymerization activity of PmHS2 and of the combined PmHS2-GlcUA⁺/PmHS2-GlcNAc⁺. The activity of PmHS2 (□) or the single action transferases incubated together (■) was determined in the presence of a 5 mm concentration of each UDP-sugar. The UDP-sugar conversion after 4 h of incubation at temperatures from 22 to 49 °C was assayed by the coupled enzyme assay; 100% activity corresponds to the highest UDP-sugar conversion observed on the temperature range. The maximal conversion was 54.0 ± 3.5 nmol/μg (□) for PmHS2 and 8.0 ± 0.7 nmol/μg (■) for PmHS2-GlcUA⁺/PmHS2-GlcNAc⁺.

FIGURE 3.

High performance anion exchange chromatography to assess PmHS2-enzyme hydrolysis activity. A, purified PmHS2 (55–60 μg/ml); B, purified single action transferases (80–90 μg/ml) (PmHS2-GlcUA⁺ or PmHS2-GlcNAc⁺) were incubated in the presence of both UDP-sugars or with only one UDP-sugar (0.25 mm). Each reaction mixture was analyzed by HPAEC (condition 1) after 24 h of incubation. Sample 1 is the inactivated PmHS2 (99 °C/15 min) incubated in the presence of UDP-GlcNAc/UDP-GlcUA. Samples 2–4 are the reaction mixtures of active PmHS2 enzymes (PmHS2, PmHS2-GlcUA⁺, or PmHS2-GlcNAc⁺) incubated with UDP-GlcNAc/UDP-GlcUA (2), UDP-GlcNAc (3), and UDP-GlcUA (4), respectively. The monosaccharides GlcNAc, GlcUA, and the UDP-GlcNAc eluted after 8, 44, and 67 min, respectively (arrows).

FIGURE 4.

High performance anion exchange chromatography analysis of the reaction mixture of each single action transferase (PmHS2-GlcUA⁺ or PmHS2-GlcNAc⁺) using electrochemical and UV detection (HPAEC condition 2). PmHS2-GlcUA⁺ (A) and PmHS2-GlcNAc⁺ (80–90 μg/ml) (B) were incubated for 24 h in the presence of 1 mm UDP-GlcUA and 1 mm UDP-GlcNAc. In the presence of PmHS2-GlcUA⁺ (A), an additional product (asterisk) eluted around 29 min; the product was detected by both electrochemical and UV detection. Using HPAEC condition 2, the degradation of UDP-GlcUA was observed, and it resulted in the formation of UMP and a degradation product (D).

FIGURE 5.

Conversion of UDP-sugars by PmHS2 in the presence of equimolar and non-equimolar UDP-sugar concentrations. The polymerization activity after 3 h of incubation was assayed by the coupled enzyme assay and expressed in percentage of UDP-sugar converted (100% corresponds to the limiting UDP-sugar concentration present at time 0). The UDP-sugar in excess was added to a 5 mm concentration and the limiting UDP-sugar to a 0.10–2.50 mm concentration.

FIGURE 6.

PmHS2 polymerization process in the presence of “heparosan template.” Heparosan template of 35 kDa (polydispersity index = 1.17) was added to the PmHS2 (55–60 μg/ml) polymerization reaction in the presence of equimolar and non-equimolar UDP-sugar concentrations (0.25 and 5 mm). A, 2% agarose gel electrophoresis analysis of reaction mixtures after 3 and 24 h of incubation. T, heparosan template (10-fold concentrated). Reference, polymerization reaction in the absence of template and with a 5 mm concentration of each UDP-sugar. For the polymerization reactions incubated in the presence of template, sample GlcUA was obtained with an excess of UDP-GlcUA, GlcNAc with an excess of UDP-GlcNAc, and [High] with a high concentration (5 mm) and [Low] with a low concentration (0.25 mm) of each UDP-sugar, respectively. B, the corresponding reaction mixtures (3 h of incubation) were also analyzed by HPSEC reaction mixtures incubated with template and an excess of UDP-GlcNAc (A) (30 and 75 kDa), an excess of UDP-GlcUA (B) (65 kDa), and with 5 mm (C) (25 and 65 kDa) or 0.25 mm (D) (130 kDa) of each UDP-sugar, respectively. In addition, the reaction mixtures incubated in the absence of template (E) and 1-fold concentrated template (F) were also analyzed by HPSEC.