Human liver glycerol-3-phosphate dehydrogenase (hlGPDH) catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to form glycerol 3-phosphate, using the binding energy associated with the nonreacting phosphodianion of the substrate to properly orient the enzymesubstrate complex within the active site. Herein, we report the crystal structures for unliganded, binary E•NAD, and ternary E•NAD•DHAP complexes of wild type hlGPDH, illustrating a new position of DHAP, and probe the kinetics of multiple mutant enzymes with natural and truncated substrates. Mutation of Lys120, which is positioned to donate a proton to the carbonyl of DHAP, results in similar increases in the activation barrier to hlGPDH-catlyzed reduction of DHAP and to phosphite dianion activated reduction of glycolaldehyde, illustrating that these transition states show similar interactions with the cationic K120 side chain. The K120A mutation results in a 5.3 kcal/mol transition state destabilization, and 3.0 kcal/mol of the lost transition state stabilization is rescued by 1.0 M ethylammonium cation. The 6.5 kcal/mol increase in the activation barrier observed for the D260G mutant hlGPDH-catalyzed reaction represents a 3.5 kcal/mol weakening of transition state stabilization by the K120A side chain, and a 3.0 kcal/mol weakening of the interactions with other residues. The interactions, at the enzyme active site, between the K120 side chain and the Q295 and R269 side chains was likewise examined by double mutant analyses. These results provide strong evidence that the enzyme rate acceleration is due mainly or exclusively to transition state stabilization by electrostatic interactions with polar amino acid side chains.

This publication is made publicly available in the institutional repository of Wageningen University and Research, under the terms of article 25fa of the Dutch Copyright Act, also known as the Amendment Taverne. This has been done with explicit consent by the author.Article 25fa states that the author of a short scientific work funded either wholly or partially by Dutch public funds is entitled to make that work publicly available for no consideration following a reasonable period of time after the work was first published, provided that clear reference is made to the source of the first publication of the work.This publication is distributed under The Association of Universities in the Netherlands (VSNU) 'Article 25fa implementation' project. In this project research outputs of researchers employed by Dutch Universities that comply with the legal requirements of Article 25fa of the Dutch Copyright Act are distributed online and free of cost or other barriers in institutional repositories. Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication.

Aerobactin, a citryl-hydroxamate siderophore, is produced by a number of pathogenic Gram-negative bacteria to aid in iron assimilation. Interest in this well-known siderophore was reignited by recent investigations suggesting that it plays a key role in mediating the enhanced virulence of a hypervirulent pathotype of (hvKP). In contrast to classical opportunistic strains of, hvKP causes serious life-threatening infections in previously healthy individuals in the community. Multiple contemporary reports have confirmed fears that the convergence of multidrug-resistant and hvKP pathotypes has led to the evolution of a highly transmissible, drug-resistant, and virulent "super bug." Despite hvKP harboring four distinct siderophore operons, knocking out production of aerobactin led to a significant attenuation of virulence. Herein, we continue our structural and functional studies on the biosynthesis of this crucial virulence factor. heterologous production and reconstitution of aerobactin biosynthesis from hvKP was carried out, demonstrating the specificity, stereoselectivity, and kinetic throughput of the complete pathway. Additionally, we present a steady-state kinetic analysis and the X-ray crystal structure of the second aerobactin synthetase IucC, as well as describe a surface entropy reduction strategy that was employed for structure determination. Finally, we show solution X-ray scattering data that support a unique dimeric quaternary structure for IucC. These new insights into aerobactin assembly will help inform potential antivirulence strategies and advance our understanding of siderophore biosynthesis.

Moroidin is a bicyclic plant octapeptide with tryptophan side-chain cross-links, originally isolated as a pain-causing agent from the Australian stinging tree Dendrocnide moroides. Moroidin and its analog celogentin C, derived from Celosia argentea, are inhibitors of tubulin polymerization and, thus, lead structures for cancer therapy. However, low isolation yields from source plants and challenging organic synthesis hinder moroidin-based drug development. Here, we present biosynthesis as an alternative route to moroidin-type bicyclic peptides and report that they are ribosomally synthesized and posttranslationally modified peptides (RiPPs) derived from BURP-domain peptide cyclases in plants. By mining 793 plant transcriptomes for moroidin core peptide motifs within BURP-domain precursor peptides, we identified a moroidin cyclase in Japanese kerria, which catalyzes the installation of the tryptophan-indole-centered macrocyclic bonds of the moroidin bicyclic motif in the presence of cupric ions. Based on the kerria moroidin cyclase, we demonstrate the feasibility of producing diverse moroidins including celogentin C in transgenic tobacco plants and report specific cytotoxicity of celogentin C against a lung adenocarcinoma cancer cell line. Our study sets the stage for future biosynthetic development of moroidin-based therapeutics and highlights that mining plant transcriptomes can reveal bioactive cyclic peptides and their underlying cyclases from new source plants.