Activation mechanism and activity of globupain, a thermostable C11 protease from the Arctic Mid-Ocean Ridge hydrothermal system

Abstract

Deep-sea hydrothermal vents offer unique habitats for heat tolerant enzymes with potential new enzymatic properties. Here, we present the novel C11 protease globupain, which was prospected from a metagenome-assembled genome of uncultivated Archaeoglobales sampled from the Soria Moria hydrothermal vent system located on the Arctic Mid-Ocean Ridge. Sequence comparisons against the MEROPS-MPRO database showed that globupain has the highest sequence identity to C11-like proteases present in human gut and intestinal bacteria. Successful recombinant expression in Escherichia coli of the wild-type zymogen and 13 mutant substitution variants allowed assessment of residues involved in maturation and activity of the enzyme. For activation, globupain required the addition of DTT and Ca²⁺. When activated, the 52kDa proenzyme was processed at K₁₃₇ and K₁₄₄ into a 12kDa light- and 32kDa heavy chain heterodimer. A structurally conserved H₁₃₂/C₁₈₅ catalytic dyad was responsible for the proteolytic activity, and the enzyme demonstrated the ability to activate in-trans. Globupain exhibited caseinolytic activity and showed a strong preference for arginine in the P1 position, with Boc-QAR-aminomethylcoumarin (AMC) as the best substrate out of a total of 17 fluorogenic AMC substrates tested. Globupain was thermostable (T_{m activated enzyme} = 94.51°C ± 0.09°C) with optimal activity at 75°C and pH 7.1. Characterization of globupain has expanded our knowledge of the catalytic properties and activation mechanisms of temperature tolerant marine C11 proteases. The unique combination of features such as elevated thermostability, activity at relatively low pH values, and ability to operate under high reducing conditions makes globupain a potential intriguing candidate for use in diverse industrial and biotechnology sectors.

Keywords: clostripain; cysteine peptidase; extracellular enzyme; hydrothermal vent; metagenome bioprospecting.

Figure 1

Sequence alignment of the C11 proteases globupain (Archaeoglobus), clostripain (Clostridium histolyticum), distapain (Parabacteroides distasonis), PmC11 (Parabacteroides merdae), and thetapain (Bacteroides thetaiotaomicron) by ESPript 3.0. Symbols depict results from site-directed mutagenesis of the globupain coding sequence; , His/Cys catalytic dyad; , sites showing resistance against cleavage when the amino acid was mutated into alanine; , sites able to cleave when mutated into alanine. The detected N-terminal residues following activation are shown in bold.

Figure 2

Globupain modeled structure predicted with AlphaFold in comparison to PmC11. (A) AlphaFold predicted structure of globupain is represented by cartoon with transparent surface (pale cyan). (B) AlphaFold of globupain compared to crystallized PmC11 (PDB ID: 4YEC) structure (light pink) in a 3D alignment showing their structural similarity. (C) Active site residues (e.g., His and Cys) are conserved among the two aligned structures. (D) Light-and heavy chain cleavage region is depicted for the modeled globupain superimposed to PmC11’s structure, as well as the (E) likely C-terminal cleavage region. Images were generated with PyMOL (v0.99c).

Figure 3

Activation of globupain. (A) Schematic representation of the primary structure of globupain. The enzyme was overproduced without the N-terminal 21 amino acid signal peptide (SP). The light chain (yellow) and the heavy chain (green) of the active heterodimer result from zymogen activation. Cleavage sites at K₁₃₇ and K₁₄₄ are shown. H₁₃₂ and C₁₈₅ of the catalytic dyad are indicated on the light-and heavy chain, respectively. The putative C-terminal region is indicated with gray stripes. Single and double mutation variants of K₃₈₃ and R₃₉₆ were tested for zymogen activation. (B) SDS-PAGE gel presentation of inactive C₁₈₅A and wild-type (WT) of 52 kDa, respectively, and activation of WT into a 32 kDa heavy-and 12 kDa light chain when incubated at 75°C for 4.5 h in activation buffer. The region within the red rectangle was excised for N-terminal sequencing. (C) SDS-PAGE gel analysis shows that the C₁₈₅A variant and K₁₃₇A/K₁₄₄A variant cannot process into a heterodimer. (D) SDS-PAGE image of WT, C₁₈₅A and WT + C₁₈₅A incubated at 75°C for 0 h and 8 h in activation buffer shows that the enzyme is able to in-trans activate. (E) When activated for 4.5 h at 75°C in activation buffer, globupain can cleave casein whereas mutation variants H₁₃₂A, C₁₈₅A and K₁₃₇A/K₁₄₄A showed no increase in fluorescence (RFU) when assayed with EnzChek™ Protease Assay Kit at 60°C. (F) Casein-gelzan™ plate showing globupain zymogen and clearance zones when activated at 75°C for 0–8 h in activation buffer.

Figure 4

Substrate utilization by globupain. WT enzyme was assayed against 50 μM of each fluorescent substrate at 50°C, fluorescence was measured, and the rate of enzyme cleavage, V_max, for each substrate is reported. (A) Globupain was assayed against the substrates initially designed for PmC11. (B) Globupain was assayed against 7 additional substrates with Lys at P1. (C) Globupain was assayed against 7 additional substrates with Arg at P1.

Figure 5

Thermoactivity and thermostability of WT globupain. (A) Optimal temperature of globupain activity determined by incubating globupain with the lead substrate, Boc-QAR-AMC, at various temperatures and inactivating with urea before measuring fluorescence which correlated to substrate cleavage. (B) Thermogram of zymogen and activated form of globupain. Y-axis represents the first derivative of fluorescence intensity ratio 350/330 nm measured by nanoDSF. T_m values are the mean and standard deviation from 3 replicate measurements.

Figure 6

Effect of pH on activity and autolysis of globupain. (A) pH optimum resolved by assaying with the substrate Boc-QAR-AMC in buffers ranging from pH 2 to pH 8. Enzyme activity is shown as V_max for the different pH-values. (B) Time-dependent loss of enzyme activity at pH 5.5 and 7.1, respectively. Activity is shown as relative percent with standard deviations based on RFU measurements. (C) SDS-PAGE gel presentation reveals intact globupain after incubation at pH 5.5 whereas at pH 7.1 (D), autolysis is observed, explaining the loss of activity in (B).