The Major Protein Arginine Methyltransferase in Trypanosoma brucei Functions as an Enzyme-Prozyme Complex

Abstract

Edited by John M. DenuProzymes are catalytically inactive enzyme paralogs that dramatically stimulate the function of weakly active enzymes through complex formation. The two prozymes described to date reside in the polyamine biosynthesis pathway of the human parasite Trypanosoma brucei, an early branching eukaryote that lacks transcriptional regulation and regulates its proteome through posttranscriptional and posttranslational means. Arginine methylation is a common posttranslational modification in eukaryotes catalyzed by protein arginine methyltransferases (PRMTs) that are typically thought to function as homodimers. We demonstrate that a major T. brucei PRMT, TbPRMT1, functions as a heterotetrameric enzyme-prozyme pair. The inactive PRMT paralog, TbPRMT1 PRO , is essential for catalytic activity of the TbPRMT1 ENZ subunit. Mutational analysis definitively demonstrates that TbPRMT1 ENZ is the cofactor-binding subunit and carries all catalytic activity of the complex. Our results are the first demonstration of an obligate heteromeric PRMT, and they suggest that enzyme-prozyme organization is expanded in trypanosomes as a posttranslational means of enzyme regulation.

The N‐terminal methyltransferase homologs NRMT1 and NRMT2 exhibit novel regulation of activity through heterotrimer formation

“…These data indicate that, while overall unique, the interaction between NRMT1 and NRMT2 has the most in common with the interactions of PRMT1/3 and METTL3/14. Like PRMT1/3 and METTL3/14, binding of the homologs increases the stability of the active enzyme . Similar to METTL14, but unlike PRMT3, NRMT2 has weak catalytic activity in vitro .…”

“…Like PRMT1/3 and METTL3/14, binding of the homologs increases the stability of the active enzyme. 4,7,9 Similar to METTL14, but unlike PRMT3, NRMT2 has weak catalytic activity in vitro. 4,7,9 However, SAM was visualized in the active site of the NRMT2 crystal, and we have seen that CRISPR-mediated knockout of NRMT1 results in a complete loss of N-terminal trimethylation, but N-terminal monomethylation remains the same [ Fig.…”

“…A common theme emerging is complex formation between methyltransferase homologs. In Trypanosoma brucei , the major type I arginine methyltransferase, PRMT1, has a very narrow substrate specificity and weak activity in vitro . However, co‐expression with its close paralog PRMT3 increases PRMT1 stability, and subsequently, its activity .…”

“…In Trypanosoma brucei , the major type I arginine methyltransferase, PRMT1, has a very narrow substrate specificity and weak activity in vitro . However, co‐expression with its close paralog PRMT3 increases PRMT1 stability, and subsequently, its activity . PRMT3 is missing key catalytic residues and does not exhibit in vitro enzymatic activity and has been characterized as a prozyme .…”

“…4 PRMT3 is missing key catalytic residues and does not exhibit in vitro enzymatic activity and has been characterized as a prozyme. 4 Activation of a catalytically weak enzyme by an inactive prozyme paralog has also been seen in T. brucei polyamine biosynthesis and described as the prozyme paradigm. 5 On average, the enzyme/prozyme complex typically exceeds the activity of the active enzyme alone by 2000-fold.…”