Correction MEDICAL SCIENCES Correction for “Regulation of T cell activation, anxiety, and male aggression by RGS2,” by Antonio J. Oliveira-dos-Santos, Goichi Matsumoto, Bryan E. Snow, Donglin Bai, Frank P. Houston, Ian Q. Whishaw, Sanjeev Mariathasan, Takehiko Sasaki, Andrew Wakeham, Pamela S. Ohashi, John C. Roder, Carol A. Barnes, David P. Siderovski, and Josef M. Penninger, which was first published October 10, 2000; 10.1073/pnas.220414397 (Proc. Natl. Acad. Sci. U.S.A. 97, 12272–12277). The authors note, “Fig. 2 contains a mistake in panel A as the + − − − CD4/CD8 scatterplots for rgs2 / and rgs2 / mice were dupli- cated when preparing the figure for publication. Multiple inde- pendent experiments showed and confirmed that there is no difference between the groups in the thymocyte populations. Therefore none of the conclusions of the paper are affected. We have included a new set of graphs for Fig 2A from an indepen- dent experiment performed concurrently with that in the pub- lished paper. We apologize for the oversight in preparing the original figure.” The revised figure and its legend appear below. − − Fig. 2. Impaired proliferation and IL-2 production by rgs2 / T cells. (A) + + + Normal populations of CD4 and CD8 T cells and B220 B cells in lymph − − nodes of rgs2 / mice. Numbers in each quadrant represent percentages of each subset. Published under the PNAS license. First published September 21, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2018014117 25182 | PNAS | October 6, 2020 | vol. 117 | no. 40 www.pnas.org Downloaded by guest on September 27, 2021 Correction BIOCHEMISTRY, CHEMISTRY Correction for “Genomic analysis of siderophore β-hydroxylases reveals divergent stereocontrol and expands the condensation domain family,” by Zachary L. Reitz, Clifford D. Hardy, Jaewon Suk, Jean Bouvet, and Alison Butler, which was first published September 16, 2019; 10.1073/pnas.1903161116 (Proc. Natl. Acad. Sci. U.S.A. 116, 19805–19814). The authors note that the grant number NSF CHE-171076 should instead appear as NSF CHE-1710761. Published under the PNAS license. First published September 21, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2018013117 CORRECTION www.pnas.org PNAS | September 29, 2020 | vol. 117 | no. 39 | 24599 Genomic analysis of siderophore β-hydroxylases reveals divergent stereocontrol and expands the condensation domain family Zachary L. Reitza, Clifford D. Hardya, Jaewon Suka, Jean Bouveta, and Alison Butlera,1 aDepartment of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106-9510 Edited by J. Martin Bollinger Jr., The Pennsylvania State University, University Park, PA, and accepted by Editorial Board Member Stephen J. Benkovic August 23, 2019 (received for review February 22, 2019) Genome mining of biosynthetic pathways streamlines discovery of corrugatin, ornicorrugatin, or histicorrugatin (SI Appendix,Fig.S1), secondary metabolites but can leave ambiguities in the predicted which contain both β-OHHis and β-OHAsp (9–11). One structures, which must be rectified experimentally. Through coupling β-hydroxyasparagine–containing siderophore has been reported, the reactivity predicted by biosynthetic gene clusters with verified pyoverdine VLB120 from Pseudomonas taiwanensis VLB120 (SI structures, the origin of the β-hydroxyaspartic acid diastereomers in Appendix,Fig.S1) (12, 13). siderophores is reported herein. Two functional subtypes of non- Peptidic siderophores are synthesized by large, multidomain heme Fe(II)/α-ketoglutarate–dependent aspartyl β-hydroxylases are nonribosomal peptide synthetase (NRPS) enzymes (14). The core identified in siderophore biosynthetic gene clusters, which differ in domains of a NRPS are adenylation (A), thiolation (T), and — β genomic organization existing either as fused domains (I HAsp)at condensation (C). The catalytic cycle begins with the activation of the carboxyl terminus of a nonribosomal peptide synthetase (NRPS) a select carboxylic acid by the A domain; this substrate, most often β — or as stand-alone enzymes (T HAsp) and each directs opposite an L-amino acid, can be predicted by sequence analysis (15). After stereoselectivity of Asp β-hydroxylation. The predictive power of this activation, the acid adenylate is transferred to the phosphopante- subtype delineation is confirmed by the stereochemical characteriza- theine (Ppant) tether of the T domain. Each peptide bond is tion of β-OHAsp residues in pyoverdine GB-1, delftibactin, histicorru- formed by a C domain, which condenses 2 T-bound substrates. CHEMISTRY gatin, and cupriachelin. The L-threo (2S,3S) β-OHAsp residues of The growing peptide chain travels down the NRPS “assembly β alterobactin arise from hydroxylation by the -hydroxylase domain line,” and the final peptide is released at the C terminus of the erythro S R β integrated into NRPS AltH, while L- (2 ,3 ) -OHAsp in delfti- NRPS, commonly by a thioesterase (Te) domain. Peptide bond- β bactin arises from the stand-alone -hydroxylase DelD. Cupriachelin forming C domains are canonical members of the condensation threo erythro β contains both L- and L- -OHAsp, consistent with the domain superfamily, which also contains other NRPS domains that β presence of both types of -hydroxylases in the biosynthetic gene catalyze diverse reactions (16, 17). The most well studied is the α – cluster. A third subtype of nonheme Fe(II)/ -ketoglutarate depen- epimerization (E) domain, which catalyzes the conversion of an β L threo dent enzymes (I H ) hydroxylates histidyl residues with - BIOCHEMISTRY His L-amino acid to a D-amino acid. Other tailoring domains found stereospecificity. A previously undescribed, noncanonical member in NRPS enzymes are responsible for formylation, halogenation, of the NRPS condensation domain superfamily is identified, named the interface domain, which is proposed to position the β-hydroxylase and the NRPS-bound amino acid prior to hydroxylation. Through Significance mapping characterized β-OHAsp diastereomers to the phyloge- netic tree of siderophore β-hydroxylases, methods to predict Bacteria produce siderophores to sequester iron(III). Genome β-OHAsp stereochemistry in silico are realized. mining of nonribosomal peptide synthetases predicts partial structures of peptidic siderophores; however, current tools cannot siderophore | hydroxyaspartic acid | biosynthesis | genomics | reliably predict which aspartate and histidine residues will be nonribosomal peptide synthetase hydroxylated to form bidentate chelating groups, nor the result- ing stereochemistry. We identified 2 functional subtypes of non- heme Fe(II)/α-ketoglutarate–dependent aspartyl β-hydroxylases in he vast majority of life requires iron as an enzyme cofactor. siderophore biosynthetic gene clusters and one type of histidyl Because Fe(III) is nearly insoluble under aerobic physiological T β-hydroxylase. Stand-alone genes encode one class of aspartyl conditions, bacteria have evolved a variety of mechanisms to meet β-hydroxylases and the histidyl β-hydroxylases, while the second their metabolic iron needs, including the biosynthesis of small- class of aspartyl β-hydroxylases is encoded within a domain of a molecule, high-affinity iron chelators called siderophores (1). Some nonribosomal peptide synthetase (NRPS) gene. Each aspartyl peptidic siderophores contain β-hydroxyaspartate (β-OHAsp), β-hydroxylase subtype effects distinct diastereoselectivity. Map- which provides bidentate OO′ coordination to Fe(III) (2). The first ping the β-OHAsp diastereomers in siderophores to the phylo- structural determination of a β-OHAsp–containing siderophore genetic tree of β-hydroxylases enables prediction of β-OHAsp came with the crystallization of ferric pyoverdine from Pseudo- stereochemistry in silico. monas B10 (SI Appendix, Fig. S1) in 1981 (3). Since then, a variety β of peptidic siderophores with -OHAsp have been character- Author contributions: Z.L.R., C.D.H., and A.B. designed research; Z.L.R., C.D.H., J.S., and ized from both marine and terrestrial bacteria. Like other J.B. performed research; Z.L.R., C.D.H., J.S., J.B., and A.B. analyzed data; and Z.L.R. and α-hydroxycarboxylate ligands, β-OHAsp bound to Fe(III) can un- A.B. wrote the paper. dergo photoinduced reduction of Fe(III) to Fe(II) accompanied by The authors declare no conflict of interest. oxidative decarboxylation of the ligand (2, 4). This article is a PNAS Direct Submission. J.M.B. is a guest editor invited by the Far fewer siderophores contain the chelating group Editorial Board. β-hydroxyhistidine (β-OHHis). The first reported example is Published under the PNAS license. pyoverdine pf244 of Pseudomonas fluorescens 244 (SI Appendix, 1To whom correspondence may be addressed. Email: [email protected]. Fig. S1) (5). β-OHHis has since been identified in the peptide of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. pyoverdines from a variety of pseudomonads (6–8). Some Pseu- 1073/pnas.1903161116/-/DCSupplemental. domonas strains produce the fatty-acyl peptidic siderophores www.pnas.org/cgi/doi/10.1073/pnas.1903161116 PNAS Latest Articles | 1of10 methylation, oxidation, and reduction (14). These modifications all ylated in cupriachelin, serobactin, pacifibactin, and alterobactin add to the structural diversity of nonribosomal peptides. (Figs. 1 and 2 and SI Appendix,Fig.S1) (25, 30, 34). Several families of enzymes are responsible for the β-hydroxy Further complicating structural predictions, β-OHAsp has amino acids found in nonribosomal peptides, namely Fe-heme 2 stereocenters