The northern corn leaf blight resistance gene Ht1 encodes an nucleotide-binding, leucine-rich repeat immune receptor.

Northern corn leaf blight, caused by the fungal pathogen Exserohilum turcicum, is a major disease of maize. The first major locus conferring resistance to E. turcicum race 0, Ht1, was identified over 50 years ago, but the underlying gene has remained unknown. We employed a map-based cloning strategy to identify the Ht1 causal gene, which was found to be a coiled-coil nucleotide-binding, leucine-rich repeat (NLR) gene, which we named PH4GP-Ht1. Transgenic testing confirmed that introducing the native PH4GP-Ht1 sequence to a susceptible maize variety resulted in resistance to E. turcicum race 0. A survey of the maize nested association mapping genomes revealed that susceptible Ht1 alleles had very low to no expression of the gene. Overexpression of the susceptible B73 allele, however, did not result in resistant plants, indicating that sequence variations may underlie the difference between resistant and susceptible phenotypes. Modelling of the PH4GP-Ht1 protein indicated that it has structural homology to the Arabidopsis NLR resistance gene ZAR1, and probably forms a similar homopentamer structure following activation. RNA sequencing data from an infection time course revealed that 1 week after inoculation there was a threefold reduction in fungal biomass in the PH4GP-Ht1 transgenic plants compared to wild-type plants. Furthermore, PH4GP-Ht1 transgenics had significantly more inoculation-responsive differentially expressed genes than wild-type plants, with enrichment seen in genes associated with both defence and photosynthesis. These results demonstrate that the NLR PH4GP-Ht1 is the causal gene underlying Ht1, which represents a different mode of action compared to the previously reported wall-associated kinase northern corn leaf blight resistance gene Htn1/Ht2/Ht3.

A new family of CRISPR-type V nucleases with C-rich PAM recognition.

Most CRISPR-type V nucleases are stimulated to cleave double-stranded (ds) DNA targets by a T-rich PAM, which restricts their targeting range. Here, we identify and characterize a new family of type V RNA-guided nuclease, Cas12l, that exclusively recognizes a C-rich (5'-CCY-3') PAM. The organization of genes within its CRISPR locus is similar to type II-B CRISPR-Cas9 systems, but both sequence analysis and functional studies establish it as a new family of type V effector. Biochemical experiments show that Cas12l nucleases function optimally between 37 and 52°C, depending on the ortholog, and preferentially cut supercoiled DNA. Like other type V nucleases, it exhibits collateral nonspecific ssDNA and ssRNA cleavage activity that is triggered by ssDNA or dsDNA target recognition. Finally, we show that one family member, Asp2Cas12l, functions in a heterologous cellular environment, altogether, suggesting that this new group of CRISPR-associated nucleases may be harnessed as genome editing reagents.

Arabidopsis Carboxylesterase 20 Binds Strigolactone and Increases Branches and Tillers When Ectopically Expressed in Arabidopsis and Maize.

Severe drought stress can delay maize silk emergence relative to the pollen shedding period, resulting in poor fertilization and reduced grain yield. Methods to minimize the delay in silking could thus improve yield stability. An Arabidopsis enhancer-tagged carboxylesterase 20 (AtCXE20) line was identified in a drought tolerance screen. Ectopic expression of AtCXE20 in Arabidopsis and maize resulted in phenotypes characteristic of strigolactone (SL)-deficient mutants, including increased branching and tillering, decreased plant height, delayed senescence, hyposensitivity to ethylene, and reduced flavonols. Maize silk growth was increased by AtCXE20 overexpression, and this phenotype was partially complemented by exogenous SL treatments. In drought conditions, the transgenic maize plants silked earlier than controls and had decreased anthesis-silking intervals. The purified recombinant AtCXE20 protein bound SL in vitro, as indicated by SL inhibiting AtCXE20 esterase activity and altering AtCXE20 intrinsic fluorescence. Homology modeling of the AtCXE20 three-dimensional (3D) protein structure revealed a large hydrophobic binding pocket capable of accommodating, but not hydrolyzing SLs. The AtCXE20 protein concentration in transgenic maize tissues was determined by mass spectrometry to be in the micromolar range, well-above known endogenous SL concentrations. These results best support a mechanism where ectopic expression of AtCXE20 with a strong promoter effectively lowers the concentration of free SL by sequestration. This study revealed an agriculturally important role for SL in maize silk growth and provided a new approach for altering SL levels in plants.

History of safe exposure and bioinformatic assessment of phosphomannose-isomerase (PMI) for allergenic risk.

Newly expressed proteins in genetically engineered crops are evaluated for potential cross reactivity to known allergens as part of their safety assessment. This assessment uses a weight-of-evidence approach. Two key components of this allergenicity assessment include any history of safe human exposure to the protein and/or the source organism from which it was originally derived, and bioinformatic analysis identifying amino acid sequence relatedness to known allergens. Phosphomannose-isomerase (PMI) has been expressed in commercialized genetically engineered (GE) crops as a selectable marker since 2010 with no known reports of allergy, which supports a history of safe exposure, and GE events expressing the PMI protein have been approved globally based on expert safety analysis. Bioinformatic analyses identified an eight-amino-acid contiguous match between PMI and a frog parvalbumin allergen (CAC83047.1). While short amino acid matches have been shown to be a poor predictor of allergen cross reactivity, most regulatory bodies require such matches be assessed in support of the allergenicity risk assessment. Here, this match is shown to be of negligible risk of conferring cross reactivity with known allergens.

A catalogue of biochemically diverse CRISPR-Cas9 orthologs.

Bacterial Cas9 nucleases from type II CRISPR-Cas antiviral defence systems have been repurposed as genome editing tools. Although these proteins are found in many microbes, only a handful of variants are used for these applications. Here, we use bioinformatic and biochemical analyses to explore this largely uncharacterized diversity. We apply cell-free biochemical screens to assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of 79 Cas9 proteins, thus identifying at least 7 distinct gRNA classes and 50 different PAM sequence requirements. PAM recognition spans the entire spectrum of T-, A-, C-, and G-rich nucleotides, from single nucleotide recognition to sequence strings longer than 4 nucleotides. Characterization of a subset of Cas9 orthologs using purified components reveals additional biochemical diversity, including both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for long stretches of homology between gRNA and DNA target. Our results expand the available toolset of RNA-programmable CRISPR-associated nucleases.

Template-based peptide modeling for celiac risk assessment of newly expressed proteins in GM crops.

Newly expressed proteins in genetically modified (GM) crops are subject to celiac disease risk assessment according to EFSA guidelines. Amino acid identity matches between short peptides (9aa) and known celiac restricted epitopes are required to be further evaluated through peptide modeling; however, validated methods and criteria are not yet available. In this investigation, several structures of HLA-DQ2.5/peptide/TCR (T-cell receptor) complexes were analyzed and two template-based peptide molding software packages were evaluated using various peptides including ones not associated with celiac disease. Structural characterization indicates that residues at P(position)1, P2, P5, P8, and P9 in the 9aa restricted epitopes also contribute to the binding of celiac peptides to the HLA-DQ2.5 antigen in addition to the presence of the motif Q/EX1PX2 starting at P4 or P6. The recognition of the HLA-DQ2.5/peptide complex by TCR is through specific interactions between the residues in the restricted epitopes and some loop structures in the TCR. The template-based software package GalaxyPepDock seems to be suitable for the application of peptide modeling when an estimated accuracy value of >0.95 combined with >160 interaction similarity score are used as a threshold for biologically meaningful in silico binding. Nevertheless, caution should be exercised when applying peptide modeling to celiac disease risk assessment until methods are rigorously validated and further evaluated to demonstrate its value in the risk assessment of newly expressed proteins in GM crops.

PAM recognition by miniature CRISPR-Cas12f nucleases triggers programmable double-stranded DNA target cleavage.

In recent years, CRISPR-associated (Cas) nucleases have revolutionized the genome editing field. Being guided by an RNA to cleave double-stranded (ds) DNA targets near a short sequence termed a protospacer adjacent motif (PAM), Cas9 and Cas12 offer unprecedented flexibility, however, more compact versions would simplify delivery and extend application. Here, we present a collection of 10 exceptionally compact (422-603 amino acids) CRISPR-Cas12f nucleases that recognize and cleave dsDNA in a PAM dependent manner. Categorized as class 2 type V-F, they originate from the previously identified Cas14 family and distantly related type V-U3 Cas proteins found in bacteria. Using biochemical methods, we demonstrate that a 5' T- or C-rich PAM sequence triggers dsDNA target cleavage. Based on this discovery, we evaluated whether they can protect against invading dsDNA in Escherichia coli and find that some but not all can. Altogether, our findings show that miniature Cas12f nucleases can protect against invading dsDNA like much larger class 2 CRISPR effectors and have the potential to be harnessed as programmable nucleases for genome editing.

Fertility restoration of maize CMS-C altered by a single amino acid substitution within the Rf4 bHLH transcription factor.

Type C cytoplasmic male sterility (CMS-C) is the most commonly used form of CMS in maize hybrid seed production. Restorer of fertility 4 (Rf4), the major fertility restorer gene of CMS-C, is located on chromosome 8S. To positionally clone Rf4, a large F3 population derived from a cross between a non-restorer and restorer (n = 5104) was screened for recombinants and then phenotyped for tassel fertility, resulting in a final map-based cloning interval of 12 kb. Within this 12-kb interval, the only likely candidate for Rf4 was GRMZM2G021276, a basic helix-loop-helix (bHLH) transcription factor with tassel-specific expression. The Rf4 gene product contains a nuclear localization signal and is likely to not interact directly with the mitochondria. Sequence analysis of Rf4 revealed four encoded amino acid substitutions between restoring and non-restoring inbreds, however only one substitution, F187Y, was within the highly conserved bHLH domain. The hypothesis that Rf4 restoration is altered by a single amino acid was tested by using clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated protein 9 (Cas9) homology directed repair (HDR) to create isogenic lines that varied for the F187Y substitution. In a population of these CRISPR-Cas9 edited plants (n = 780) that was phenotyped for tassel fertility, plants containing F187 were completely fertile, indicating fertility restoration, and plants containing Y187 were sterile, indicating lack of fertility restoration. Structural modeling shows that this amino acid residue 187 is located within the four helix bundle core, a critical region for stabilizing dimer conformation and affecting interaction partner selection.

Overexpression of zmm28 increases maize grain yield in the field.

Increasing maize grain yield has been a major focus of both plant breeding and genetic engineering to meet the global demand for food, feed, and industrial uses. We report that increasing and extending expression of a maize MADS-box transcription factor gene, zmm28, under the control of a moderate-constitutive maize promoter, results in maize plants with increased plant growth, photosynthesis capacity, and nitrogen utilization. Molecular and biochemical characterization of zmm28 transgenic plants demonstrated that their enhanced agronomic traits are associated with elevated plant carbon assimilation, nitrogen utilization, and plant growth. Overall, these positive attributes are associated with a significant increase in grain yield relative to wild-type controls that is consistent across years, environments, and elite germplasm backgrounds.

Identification and characterization of a novel stay-green QTL that increases yield in maize.

Functional stay-green is a valuable trait that extends the photosynthetic period, increases source capacity and biomass and ultimately translates to higher grain yield. Selection for higher yields has increased stay-green in modern maize hybrids. Here, we report a novel QTL controlling functional stay-green that was discovered in a mapping population derived from the Illinois High Protein 1 (IHP1) and Illinois Low Protein 1 (ILP1) lines, which show very different rates of leaf senescence. This QTL was mapped to a single gene containing a NAC-domain transcription factor that we named nac7. Transgenic maize lines where nac7 was down-regulated by RNAi showed delayed senescence and increased both biomass and nitrogen accumulation in vegetative tissues, demonstrating NAC7 functions as a negative regulator of the stay-green trait. More importantly, crosses between nac7 RNAi parents and two different elite inbred testers produced hybrids with prolonged stay-green and increased grain yield by an average 0.29 megagram/hectare (4.6 bushel/acre), in 2 years of multi-environment field trials. Subsequent RNAseq experiments, one employing nac7 RNAi leaves and the other using leaf protoplasts overexpressing Nac7, revealed an important role for NAC7 in regulating genes in photosynthesis, chlorophyll degradation and protein turnover pathways that each contribute to the functional stay-green phenotype. We further determined the putative target of NAC7 and provided a logical extension for the role of NAC7 in regulating resource allocation from vegetative source to reproductive sink tissues. Collectively, our findings make a compelling case for NAC7 as a target for improving functional stay-green and yields in maize and other crops.

Desensitizing plant EPSP synthase to glyphosate: Optimized global sequence context accommodates a glycine-to-alanine change in the active site.

5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the transfer of a carboxyvinyl group from phosphoenolpyruvate (PEP) to shikimate-3-phosphate and in plants is the target of the herbicide glyphosate. EPSPSs with high catalytic efficiency and insensitivity to glyphosate are of microbial origin, including the enzyme from Agrobacterium strain CP4, in which insensitivity is conferred by an active site alanine. In the sequence context of plant EPSPSs, alanine in place of glycine at the equivalent position interferes with the binding of both glyphosate and PEP. We show here that iterative optimization of maize EPSPS containing the G101A substitution yielded variants on par with CP4 in terms of catalytic activity in the presence of glyphosate. The improvement relative to G101A alone was entirely due to reduction in Km for PEP from 333 to 18 µm, versus 9.5 µm for native maize EPSPS. A large portion of the reduction in Km was conferred by two down-sizing substitutions (L97C and V332A) within 8 Å of glyphosate, which together reduced Km for PEP to 43 µm Although the original optimization was conducted with maize EPSPS, contextually homologous substitutions conferred similar properties to the EPSPSs of other crops. We also discovered a variant having the known glyphosate-desensitizing substitution P106L plus three additional ones that reduced the Km for PEP from 47 µm, observed with P106L alone, to 10.3 µm The improvements obtained with both Ala101 and Leu106 have implications regarding glyphosate-tolerant crops and weeds.

An Enterotoxin-Like Binary Protein from Pseudomonas protegens with Potent Nematicidal Activity.

Soil microbes are a major food source for free-living soil nematodes. It is known that certain soil bacteria have evolved systems to combat predation. We identified the nematode-antagonistic Pseudomonas protegens strain 15G2 from screening of microbes. Through protein purification we identified a binary protein, designated Pp-ANP, which is responsible for the nematicidal activity. This binary protein inhibits Caenorhabditis elegans growth and development by arresting larvae at the L1 stage and killing older-staged worms. The two subunits, Pp-ANP1a and Pp-ANP2a, are active when reconstituted from separate expression in Escherichia coli The binary toxin also shows strong nematicidal activity against three other free-living nematodes (Pristionchus pacificus, Panagrellus redivivus, and Acrobeloides sp.), but we did not find any activity against insects and fungi under test conditions, indicating specificity for nematodes. Pp-ANP1a has no significant identity to any known proteins, while Pp-ANP2a shows ∼30% identity to E. coli heat-labile enterotoxin (LT) subunit A and cholera toxin (CT) subunit A. Protein modeling indicates that Pp-ANP2a is structurally similar to CT/LT and likely acts as an ADP-ribosyltransferase. Despite the similarity, Pp-ANP shows several characteristics distinct from CT/LT toxins. Our results indicate that Pp-ANP is a new enterotoxin-like binary toxin with potent and specific activity to nematodes. The potency and specificity of Pp-ANP suggest applications in controlling parasitic nematodes and open an avenue for further research on its mechanism of action and role in bacterium-nematode interaction.IMPORTANCE This study reports the discovery of a new enterotoxin-like binary protein, Pp-ANP, from a Pseudomonas protegens strain. Pp-ANP shows strong nematicidal activity against Caenorhabditis elegans larvae and older-staged worms. It also shows strong activity on other free-living nematodes (Pristionchus pacificus, Panagrellus redivivus, and Acrobeloides sp.). The two subunits, Pp-ANP1a and Pp-ANP2a, can be expressed separately and reconstituted to form the active complex. Pp-ANP shows some distinct characteristics compared with other toxins, including Escherichia coli enterotoxin and cholera toxin. The present study indicates that Pp-ANP is a novel binary toxin and that it has potential applications in controlling parasitic nematodes and in studying toxin-host interaction.

A pair of homoeolog ClpP5 genes underlies a virescent yellow-like mutant and its modifier in maize.

Gene-background interaction is a commonly observed phenomenon in many species, but the molecular mechanisms of such an interaction is less well understood. Here we report the cloning of a maize mutant gene and its modifier. A recessive mutant with a virescent yellow-like (vyl) phenotype was identified in an ethyl methanesulfonate-mutagenized population derived from the maize inbred line B73. Homozygous mutant maize plants exhibited a yellow leaf phenotype after emergence but gradually recovered and became indistinguishable from wild-type plants after approximately 2 weeks. Taking the positional cloning approach, the Chr.9_ClpP5 gene, one of the proteolytic subunits of the chloroplast Clp protease complex, was identified and validated as the candidate gene for vyl. When introgressed by backcross into the maize inbred line PH09B, the mutant phenotype of vyl lasted much longer in the greenhouse and was lethal in the field, implying the presence of a modifier(s) for vyl. A major modifier locus was identified on chromosome 1, and a paralogous ClpP5 gene was isolated and confirmed as the candidate for the vyl-modifier. Expression of Chr.1_ClpP5 is induced significantly in B73 by the vyl mutation, while the expression of Chr.1_ClpP5 in PH09B is not responsive to the vyl mutation. Moreover, expression and sequence analysis suggests that the PH09B Chr.1_ClpP5 allele is functionally weaker than the B73 allele. We propose that functional redundancy between duplicated paralogous genes is the molecular mechanism for the interaction between vyl and its modifier.

Maize ARGOS1 (ZAR1) transgenic alleles increase hybrid maize yield.

Crop improvement for yield and drought tolerance is challenging due to the complex genetic nature of these traits and environmental dependencies. This study reports that transgenic over-expression of Zea mays AR GOS1 (ZAR1) enhanced maize organ growth, grain yield, and drought-stress tolerance. The ZAR1 transgene exhibited environmental interactions, with yield increase under Temperate Dry and yield reduction under Temperate Humid or High Latitude environments. Native ZAR1 allele variation associated with drought-stress tolerance. Two founder alleles identified in the mid-maturity germplasm of North America now predominate in Pioneer's modern breeding programme, and have distinct proteins, promoters and expression patterns. These two major alleles show heterotic group partitioning, with one predominant in Pioneer's female and the other in the male heterotic groups, respectively. These two alleles also associate with favourable crop performance when heterozygous. Allele-specific transgene testing showed that, of the two alleles discussed here, each allele differed in their impact on yield and environmental interactions. Moreover, when transgenically stacked together the allelic pair showed yield and environmental performance advantages over either single allele, resembling heterosis effects. This work demonstrates differences in transgenic efficacy of native alleles and the differences reflect their association with hybrid breeding performance.

Cell Number Regulator1 affects plant and organ size in maize: implications for crop yield enhancement and heterosis.

Genes involved in cell number regulation may affect plant growth and organ size and, ultimately, crop yield. The tomato (genus Solanum) fruit weight gene fw2.2, for instance, governs a quantitative trait locus that accounts for 30% of fruit size variation, with increased fruit size chiefly due to increased carpel ovary cell number. To expand investigation of how related genes may impact other crop plant or organ sizes, we identified the maize (Zea mays) gene family of putative fw2.2 orthologs, naming them Cell Number Regulator (CNR) genes. This family represents an ancient eukaryotic family of Cys-rich proteins containing the PLAC8 or DUF614 conserved motif. We focused on native expression and transgene analysis of the two maize members closest to Le-fw2.2, namely, CNR1 and CNR2. We show that CNR1 reduced overall plant size when ectopically overexpressed and that plant and organ size increased when its expression was cosuppressed or silenced. Leaf epidermal cell counts showed that the increased or decreased transgenic plant and organ size was due to changes in cell number, not cell size. CNR2 expression was found to be negatively correlated with tissue growth activity and hybrid seedling vigor. The effects of CNR1 on plant size and cell number are reminiscent of heterosis, which also increases plant size primarily through increased cell number. Regardless of whether CNRs and other cell number-influencing genes directly contribute to, or merely mimic, heterosis, they may aid generation of more vigorous and productive crop plants.

A genomic and expression compendium of the expanded PEBP gene family from maize.

The phosphatidylethanolamine-binding proteins (PEBPs) represent an ancient protein family found across the biosphere. In animals they are known to act as kinase and serine protease inhibitors controlling cell growth and differentiation. In plants the most extensively studied PEBP genes, the Arabidopsis (Arabidopsis thaliana) FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1) genes, function, respectively, as a promoter and a repressor of the floral transition. Twenty-five maize (Zea mays) genes that encode PEBP-like proteins, likely the entire gene family, were identified and named Zea mays CENTRORADIALIS (ZCN), after the first described plant PEBP gene from Antirrhinum. The maize family is expanded relative to eudicots (typically six to eight genes) and rice (Oryza sativa; 19 genes). Genomic structures, map locations, and syntenous relationships with rice were determined for 24 of the maize ZCN genes. Phylogenetic analysis assigned the maize ZCN proteins to three major subfamilies: TFL1-like (six members), MOTHER OF FT AND TFL1-like (three), and FT-like (15). Expression analysis demonstrated transcription for at least 21 ZCN genes, many with developmentally specific patterns and some having alternatively spliced transcripts. Expression patterns and protein structural analysis identified maize candidates likely having conserved gene function of TFL1. Expression patterns and interaction of the ZCN8 protein with the floral activator DLF1 in the yeast (Saccharomyces cerevisiae) two-hybrid assay strongly supports that ZCN8 plays an orthologous FT function in maize. The expression of other ZCN genes in roots, kernels, and flowers implies their involvement in diverse developmental processes.

The Arabidopsis thaliana AT PRP39-1 gene, encoding a tetratricopeptide repeat protein with similarity to the yeast pre-mRNA processing protein PRP39, affects flowering time.

Flowering is regulated by a network integrated from four major pathways, including the photoperiod, vernalization, gibberellin, and autonomous pathways. RNA processing within the autonomous pathway is well known to regulate Arabidopsis thaliana flowering time. Here we identify a novel Arabidopsis gene, designated AT PRP39-1, that affects flowering time. Based on observations that homozygous at prp39-1 plants are late flowering under both long and short days and responsive to GA and vernalization treatment, we tentatively conclude that AT PRP39-1 may represent a new component of the autonomous pathway. Consistent with previous studies on genes of the autonomous pathway, knockout of AT PRP39-1 in Arabidopsis displays an upregulation of the steady state level of FLC, and simultaneous downregulation of FT and SOC1 transcript levels in adult tissues. AT PRP39-1 encodes a tetratricopeptide repeat protein with a similarity to a yeast mRNA processing protein Prp39p, suggesting that the involvement of these tetratricopeptide repeat proteins in RNA processing is conserved among yeast, human, and plants. Structure modeling suggests that AT PRP39-1 has two TPR superhelical domains suitable for target protein binding. We discuss how AT PRP39-1 may function in the control of flowering in the context of the autonomous pathway.

delayed flowering1 Encodes a basic leucine zipper protein that mediates floral inductive signals at the shoot apex in maize.

Separation of the life cycle of flowering plants into two distinct growth phases, vegetative and reproductive, is marked by the floral transition. The initial floral inductive signals are perceived in the leaves and transmitted to the shoot apex, where the vegetative shoot apical meristem is restructured into a reproductive meristem. In this study, we report cloning and characterization of the maize (Zea mays) flowering time gene delayed flowering1 (dlf1). Loss of dlf1 function results in late flowering, indicating dlf1 is required for timely promotion of the floral transition. dlf1 encodes a protein with a basic leucine zipper domain belonging to an evolutionarily conserved family. Three-dimensional protein modeling of a missense mutation within the basic domain suggests DLF1 protein functions through DNA binding. The spatial and temporal expression pattern of dlf1 indicates a threshold level of dlf1 is required in the shoot apex for proper timing of the floral transition. Double mutant analysis of dlf1 and indeterminate1 (id1), another late flowering mutation, places dlf1 downstream of id1 function and suggests dlf1 mediates floral inductive signals transmitted from leaves to the shoot apex. This study establishes an emergent framework for the genetic control of floral induction in maize and highlights the conserved topology of the floral transition network in flowering plants.

Determinants of L-aspartate and IMP recognition in Escherichia coli adenylosuccinate synthetase.

Adenylosuccinate synthetase governs the first committed step in the de novo synthesis of AMP. Mutations of conserved residues in the synthetase from Escherichia coli reveal significant roles for Val(273) and Thr(300) in the recognition of l-aspartate, even though these residues do not or cannot hydrogen bond with the substrate. The mutation of Thr(300) to alanine increases the K(m) for l-aspartate by 30-fold. In contrast, its mutation to valine causes no more than a 4-fold increase in the K(m) for l-aspartate, while increasing k(cat) by 3-fold. Mutations of Val(273) to alanine, threonine, or asparagine increase the K(m) for l-aspartate from 15- to 40-fold, and concomitantly decrease the K(i) for dicarboxylate analogues of l-aspartate by up to 40-fold. The above perturbations are comparable with those resulting from the elimination of a hydrogen bond between the enzyme and substrate: alanine mutations of Thr(128) and Thr(129) increase the K(m) for IMP by up to 30-fold and the alanine mutation of Thr(301) abolishes catalysis supported by l-aspartate, but has no effect on catalysis supported by hydroxylamine. Structure-based mechanisms, by which the above residues influence substrate recognition, are presented.

IMP Alone Organizes the Active Site of Adenylosuccinate Synthetase from Escherichia coli.

A complete set of substrate/substrate analogs of adenylosuccinate synthetase from Escherichia coli induces dimer formation and a transition from a disordered to an ordered active site. The most striking of the ligand-induced effects is the movement of loop 40-53 by up to 9 A. Crystal structures of the partially ligated synthetase, which either combine IMP and hadacidin or IMP, hadacidin, and Mg(2+)-pyrophosphate, have ordered active sites, comparable with the fully ligated enzyme. More significantly, a crystal structure of the synthetase with IMP alone exhibits a largely ordered active site, which includes the 9 A movement of loop 40-53 but does not include conformational adjustments to backbone carbonyl 40 (Mg(2+) interaction element) and loop 298-304 (L-aspartate binding element). Interactions involving the 5'-phosphoryl group of IMP evidently trigger the formation of salt links some 30 A away. The above provides a structural basis for ligand binding synergism, effects on k(cat) due to mutations far from the site of catalysis, and the complete loss of substrate efficacy due to minor alterations of the 5'-phosphoryl group of IMP.