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Enzymes are known to adopt various conformations at different points along their catalytic cycles. Here, we present a comprehensive analysis of 15 isomorphous, high resolution crystal structures of the enzyme phosphoglucomutase from the bacterium Xanthomonas citri. The protein was captured in distinct states critical to function, including enzyme-substrate, enzyme-product, and enzyme-intermediate complexes. Key residues in ligand recognition and regions undergoing conformational change are identified and correlated with the various steps of the catalytic reaction. In addition, we use principal component analysis to examine various subsets of these structures with two goals: (1) identifying sites of conformational heterogeneity through a comparison of room temperature and cryogenic structures of the apo-enzyme and (2) a priori clustering of the enzyme-ligand complexes into functionally related groups, showing sensitivity of this method to structural features difficult to detect by traditional methods. This study captures, in a single system, the structural basis of diverse substrate recognition, the subtle impact of covalent modification, and the role of ligand-induced conformational change in this representative enzyme of the α-D-phosphohexomutase superfamily.

The enzyme serine hydroxymethyltransferase (SHMT) plays a key role in folate metabolism and is conserved in all kingdoms of life. SHMT is a pyridoxal 50 -phosphate (PLP) — dependent enzyme that catalyzes the conversion of L-serine and (6S)-tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate. Crystal structures of multiple members of the SHMT family have shown that the enzyme has a single conserved cis proline, which is located near the active site. Here, we have characterized a Pro to Ser amino acid variant (P285S) that affects this conserved cis proline in soybean SHMT8. P285S was identified as one of a set of mutations that affect the resistance of soybean to the agricultural pathogen soybean cyst nematode. We find that replacement of Pro285 by serine eliminates PLP-mediated catalytic activity of SHMT8, reduces folate binding, decreases enzyme stability, and affects the dimer-tetramer ratio of the enzyme in solution. Crystal structures at 1.9–2.2 Å resolution reveal a local reordering of the polypeptide chain that extends an α-helix and shifts a turn region into the active site. This results in a dramatically perturbed PLP-binding pose, where the ring of the cofactor is flipped by ∼180° with concomitant loss of conserved enzyme-PLP interactions. A nearby region of the polypeptide becomes disordered, evidenced by missing electron density for ∼10 residues. These structural perturbations are consistent with the loss of enzyme activity and folate binding and underscore the important role of the Pro285 cis-peptide in SHMT structure and function. [ABSTRACT FROM AUTHOR]

Tetrahydrofolate and its derivatives participate in one-carbon transfer reactions in all organisms. The cellular form of tetrahydrofolate (THF) is modified by multiple glutamate residues and polyglutamylation plays a key role in organellar and cellular folate homeostasis. In addition, polyglutamylation of THF is known to increase the binding affinity to enzymes in the folate cycle, many of which can utilize polyglutamylated THF as a substrate. Here, we use Xray crystallography to provide a high-resolution view of interactions between the enzyme serine hydroxymethyltransferase (SHMT), which provides one carbon precursors for the folate cycle, and a polyglutamylated form of THF. Our 1.7 Å crystal structure of soybean SHMT8 in complex with diglutamylated 5-formyl- THF reveals, for the first time, a structural rearrangement of a loop at the entrance to the folate binding site accompanied by the formation of novel specific interactions between the enzyme and the diglutamyl tail of the ligand. Biochemical assays show that additional glutamate moieties on the folate ligand increase both enzyme stability and binding affinity. Together these studies provide new information on SHMT structure and function and inform the design of anti-folate agents. [ABSTRACT FROM AUTHOR]

This paper describes the introduction of synchrotron-based macromolecular crystallography (MX) into an undergraduate laboratory class. An introductory 2 week experimental module on MX, consisting of four laboratory sessions and two classroom lectures, was incorporated into a senior-level biochemistry class focused on a survey of biochemical techniques, including the experimental characterization of proteins. Students purified recombinant protein samples, set up crystallization plates and flash-cooled crystals for shipping to a synchrotron. Students then collected X-ray diffraction data sets from their crystals via the remote interface of the Molecular Biology Consortium beamline (4.2.2) at the Advanced Light Source in Berkeley, CA, USA. Processed diffraction data sets were transferred back to the laboratory and used in conjunction with partial protein models provided to the students for refinement and model building. The laboratory component was supplemented by up to 2 h of lectures by faculty with expertise in MX. This module can be easily adapted for implementation into other similar undergraduate classes, assuming the availability of local crystallographic expertise and access to remote data collection at a synchrotron source.

Two amino acid variants in soybean serine hydroxymethyltransferase 8 (SHMT8) are associated with resistance to the soybean cyst nematode (SCN), a devastating agricultural pathogen with worldwide economic impacts on soybean production. SHMT8 is a cytoplasmic enzyme that catalyzes the pyridoxal 5‐phosphate‐dependent conversion of serine and tetrahydrofolate (THF) to glycine and 5,10‐methylenetetrahydrofolate. A previous study of the P130R/N358Y double variant of SHMT8, identified in the SCN‐resistant soybean cultivar (cv.) Forrest, showed profound impairment of folate binding affinity and reduced THF‐dependent enzyme activity, relative to the highly active SHMT8 in cv. Essex, which is susceptible to SCN. Given the importance of SCN‐resistance in soybean agriculture, we report here the biochemical and structural characterization of the P130R and N358Y single variants to elucidate their individual effects on soybean SHMT8. We find that both single variants have reduced THF‐dependent catalytic activity relative to Essex SHMT8 (10‐ to 50‐fold decrease in kcat/Km) but are significantly more active than the P130R/N368Y double variant. The kinetic data also show that the single variants lack THF‐substrate inhibition as found in Essex SHMT8, an observation with implications for regulation of the folate cycle. Five crystal structures of the P130R and N358Y variants in complex with various ligands (resolutions from 1.49 to 2.30 Å) reveal distinct structural impacts of the mutations and provide new insights into allosterism. Our results support the notion that the P130R/N358Y double variant in Forrest SHMT8 produces unique and unexpected effects on the enzyme, which cannot be easily predicted from the behavior of the individual variants. [ABSTRACT FROM AUTHOR]

Phosphoglucomutase 1 (PGM1) plays a central role in glucose homeostasis in human cells. Missense variants of this enzyme cause an inborn error of metabolism, which is categorized as a congenital disorder of glycosylation. Here, two disease-related variants of PGM1, T337M and G391V, which are both located in domain 3 of the four-domain protein, were characterized via X-ray crystallography and biochemical assays. The studies show multiple impacts resulting from these dysfunctional variants, including both short- and long-range structural perturbations. In the T337M variant these are limited to a small shift in an active-site loop, consistent with reduced enzyme activity. In contrast, the G391V variant produces a cascade of structural perturbations, including displacement of both the catalytic phosphoserine and metal-binding loops. This work reinforces several themes that were found in prior studies of dysfunctional PGM1 variants, including increased structural flexibility and the outsized impacts of mutations affecting interdomain interfaces. The molecular mechanisms of PGM1 variants have implications for newly described inherited disorders of related enzymes. [ABSTRACT FROM AUTHOR]

ALTHOUGH THE WELL-CHARACTERized DNA binding motifs (helix-turn-helix, zinc finger, and so forth) use stably folded units of secondary structure for recognition, flexible regions can also contribute to site-specific recognition. Unfortunately, [...]

Missense variants of human phosphoglucomutase 1 (PGM1) cause the inherited metabolic disease known as PGM1 deficiency. This condition is categorised as both a glycogen storage disease and a congenital disorder of glycosylation. Approximately 20 missense variants of PGM1 are linked to PGM1 deficiency, and biochemical studies have suggested that they fall into two general categories: those affecting the active site and catalytic efficiency, and those that appear to impair protein folding and/or stability. In this study, we characterise a novel variant of Arg422, a residue distal from the active site of PGM1 and the site of a previously identified disease‐related variant (Arg422Trp). In prior studies, the R422W variant was found to produce insoluble protein in a recombinant expression system, precluding further in vitro characterisation. Here we investigate an alternative variant of this residue, Arg422Gln, which is amenable to experimental characterisation presumably due to its more conservative physicochemical substitution. Biochemical, crystallographic, and computational studies of R422Q establish that this variant causes only minor changes in catalytic efficiency and 3D structure, but is nonetheless dramatically reduced in stability. Unexpectedly, binding of a substrate analog is found to further destabilise the protein, in contrast to its stabilising effect on wild‐type PGM1 and several other missense variants. This work establishes Arg422 as a lynchpin residue for the stability of PGM1 and supports the impairment of protein stability as a pathomechanism for variants that cause PGM1 deficiency. Synopsis Biochemical and structural studies of a missense variant far from the active site of human PGM1 identify a residue with a key role in enzyme stability. [ABSTRACT FROM AUTHOR]

The site-directed mutagenesis, structural, kinetic, and thermodynamic characterization of enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) from the bacterium Pseudomonas aeruginosa is reported. The results help to better understand the backbone flexibility and conformational changes in the PMM/PGM reaction and the role of hinges in enzyme catalysis.

Research has been conducted on Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase. Results demonstrate that phosphomannomutase/phosphoglucomutase is a very elastic and resilient enzyme.

The 1.55 angsrom crystal structure of GDP-mannose dehydrogenase complexed with its cofactor NAD)H) and GDP-mannuronic acid product shows that the enzyme exhibits a domain-swapped dimer consisting of two polypeptide chains that contribute to each active site. Data indicate that the dimer interface is extensive and facilitates intersubunit communication.

The key metabolic enzyme phosphoglucomutase 1 (PGM1) controls glucose homeostasis in most human cells. Four proteins related to PGM1, known as PGM2, PGM2L1, PGM3 and PGM5, and referred to herein as paralogs, are encoded in the human genome. Although all members of the same enzyme superfamily, these proteins have distinct substrate preferences and different functional roles. The recent association of PGM1 and PGM3 with inherited enzyme deficiencies prompts us to revisit sequence-structure and other relationships among the PGM1 paralogs, which are understudied despite their importance in human biology. Using currently available sequence, structure, and expression data, we investigated evolutionary relationships, tissue-specific expression profiles, and the amino acid preferences of key active site motifs. Phylogenetic analyses indicate both ancient and more recent divergence between the different enzyme sub-groups comprising the human paralogs. Tissue-specific protein and RNA expression profiles show widely varying patterns for each paralog, providing insight into function and disease pathology. Multiple sequence alignments confirm high conservation of key active site regions, but also reveal differences related to substrate specificity. In addition, we find that sequence variants of PGM2, PGM2L1, and PGM5 verified in the human population affect residues associated with disease-related mutants in PGM1 or PGM3. This suggests that inherited diseases related to dysfunction of these paralogs will likely occur in humans. [ABSTRACT FROM AUTHOR]