Structural basis of enzyme activity regulation by the propeptide of l-lysine α-oxidase precursor from Trichoderma viride

Masaki Kitagawa,Nanako Ito,Yuya Matsumoto,Masaya Saito,Takashi Tamura,Hitoshi Kusakabe,Kenji Inagaki,Katsumi Imada

doi:10.1016/j.yjsbx.2021.100044

Abstract

Harmuful proteins are usually synthesized as inactive precursors and are activated by proteolytic processing. l-Amino acid oxidase (LAAO) is a flavoenzyme that catalyzes the oxidative deamination of l-amino acid to produce a 2-oxo acid with ammonia and highly toxic hydrogen peroxide and, therefore, is expressed as a precursor. The LAAO precursor shows significant variation in size and the cleavage pattern for activation. However, the molecular mechanism of how the propeptide suppresses the enzyme activity remains unclear except for deaminating/decarboxylating Pseudomonasl-phenylalanine oxidase (PAO), which has a short N-terminal propeptide composed of 14 residues. Here we show the inactivation mechanism of the l-lysine oxidase (LysOX) precursor (prLysOX), which has a long N-terminal propeptide composed of 77 residues, based on the crystal structure at 1.97 Å resolution. The propeptide of prLysOX indirectly changes the active site structure to inhibit the enzyme activity. prLysOX retains weak enzymatic activity with strict specificity for l-lysine and shows raised activity in acidic conditions. The structures of prLysOX crystals that soaked in a solution with various concentrations of l-lysine have revealed that prLysOX can adopt two conformations; one is the inhibitory form, and the other is very similar to mature LysOX. The propeptide region of the latter form is disordered, and l-lysine is bound to the latter form. These results indicate that prLysOX uses a different strategy from PAO to suppress the enzyme activity and suggest that prLysOX can be activated quickly in response to the environmental change without proteolytic processing.

Highlights

Many toxic or harmful proteins are expressed as inactive precursors and are activated by cleavage of specific sites and removal of the pro peptide when their function is required
Enzymatic property of the lysine oxidase (LysOX) precursor prLysOX overexpressed in E. coli was purified as previously described (Kondo et al, 2020). prLysOX showed a low specific activity of less than 1/50 of mature LysOX, suggesting that removal of the propeptide region activates LysOX
To clarify whether the removal of the propeptide in creases the enzyme activity, we examined various proteases, such as trypsin, chymotrypsin, and metalloendopeptidase from Streptomayces griseus (SGMP), to cleave off the propeptide region of prLysOX and assessed enzyme activity of the proteolytic products. prLysOX is digested to produce a stable fragment of 56 kDa, which is comparable to the molecular mass of native LysOX (Fig. 1A)

Summary

Introduction

Many toxic or harmful proteins are expressed as inactive precursors and are activated by cleavage of specific sites and removal of the pro peptide when their function is required. The role and functional mechanism of propeptide are well studied for proteases (Khan and James, 1998). X-ray structural studies of zymogens and corre sponding mature proteases have shown the structural changes induced by the cleavage of the zymogens and revealed the mechanisms of the enzyme activity regulation by the propeptides. Because LAAO produces hydrogen peroxide, which is highly toxic to living organisms, during the LAAO catalysis, most of the LAAOs are expressed as precursor proteins and are processed by proteases for activation. LAAO from Neurospora crassa synthesized as a precursor composed of 695 amino acids, and its N-terminal 129 residues are cleaved to generate the mature enzyme (566 amino acids) (Nie dermann and Lerch, 1990).

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Results

Conclusion