The G1-to-S-phase transition is a key regulatory point in the cell cycle, but the rate-limiting component in plants is unknown. Overexpression of CYCLIN D3;1 (CYCD3;1) in transgenic plants increases mitotic cycles and reduces endocycles, but its effects on cell cycle progression cannot be unambiguously determined. To analyze the cell cycle roles of plant D-type cyclins, we overexpressed CYCD3;1 in Arabidopsis thaliana cell suspension cultures. Changes in cell number and doubling time were insignificant, but cultures exhibited an increased proportion of G2-over G1-phase cells, as well as increased G2 arrest in response to stationary phase and sucrose starvation. Synchronized cultures confirm that CYCD3;1-expressing (but not CYCD2;1-expressing) cells show increased G2-phase length and delayed activation of mitotic genes such as B-type cyclins, suggesting that CYCD3;1 has a specific G1/S role. Analysis of putative cyclin-dependent kinase phosphorylation sites within CYCD3;1 shows that mutating Ser-343 to Ala enhances CYCD3;1 potency without affecting its rate of turnover and results in a fivefold increase in the level of cell death in response to sucrose removal. We conclude that CYCD3;1 dominantly drives the G1/S transition, and in sucrose-depleted cells the decline in CYCD3;1 levels leads to G1 arrest, which is overcome by ectopic CYCD3;1 expression. Ser-343 is likely a key residue in modulating CYCD3;1 activity in response to sucrose depletion.

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Aldolases are a specific group of lyases that catalyze the reversible stereoselective addition of a donor compound (nucleophile) onto an acceptor compound (electrophile). Whereas most aldolases are specific for their donor compound in the aldolization reaction, they often tolerate a wide range of aldehydes as acceptor compounds. C-C bonding by aldolases creates stereocenters in the resulting aldol products. This makes aldolases interesting tools for asymmetric syntheses of rare sugars or sugar-derived compounds as iminocyclitols, statins, epothilones, and sialic acids. Besides the well-known fructose 1,6-bisphosphate aldolase, other aldolases of microbial origin have attracted the interest of synthetic bio-organic chemists in recent years. These are either other dihydroxyacetone phosphate aldolases or aldolases depending on pyruvate/phosphoenolpyruvate, glycine, or acetaldehyde as donor substrate. Recently, an aldolase that accepts dihydroxyacetone or hydroxyacetone as a donor was described. A further enlargement of the arsenal of available chemoenzymatic tools can be achieved through screening for novel aldolase activities and directed evolution of existing aldolases to alter their substrate- or stereospecifities. We give an update of work on aldolases, with an emphasis on microbial aldolases.

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Based on a structure-assisted sequence alignment we designed 11 focused libraries at residues in the active site of transaldolase B from Escherichia coli and screened them for their ability to synthesize fructose 6-phosphate from dihydroxyacetone and glyceraldehyde 3-phosphate using a newly developed color assay. We found one positive variant exhibiting a replacement of Phe 178 to Tyr. This mutant variant is able not only to transfer a dihydroxyacetone moiety from a ketose donor, fructose 6-phosphate, onto an aldehyde acceptor, erythrose 4-phosphate (14 units/mg), but to use it as a substrate directly in an aldolase reaction (7 units/mg). With a single amino acid replacement the fructose-6-phosphate aldolase activity was increased considerably (>70-fold compared with wild-type). Structural studies of the wild-type and mutant protein suggest that this is due to a different H-bond pattern in the active site leading to a destabilization of the Schiff base intermediate. Furthermore, we show that a homologous replacement has a similar effect in the human transaldolase Taldo1 (aldolase activity, 14 units/mg). We also demonstrate that both enzymes TalB and Taldo1 are recognized by the same polyclonal antibody. Transaldolase (Tal)2 is a ubiquitous enzyme that is present in all domains of life. It is part of the non-oxidative path of the pentose phosphate pathway. Here, it catalyzes the reversible transfer of a dihydroxyacetone (DHA) moiety from a ketose donor, e.g. fructose 6-phosphate (Fru-6-P), onto an aldehyde acceptor, e.g. erythrose 4-phosphate (Ery-4-P). The best studied example is transaldolase B (TalB) from Escherichia coli (1). A number of structural and mechanistic studies have been published elucidating its reaction mechanism (1-6). Similar to class I aldolases the reaction proceeds via a Schiff base intermediate. TalB is a homo-dimer and the monomer exhibits a (␤/␣) 8 -barrel fold where the C-terminal helix lies across the barrel opening at one site (5). A similar structure had been determined for the human transaldolase (7).Recently, a fructose-6-phosphate aldolase (FSA) of E. coli has been discovered by our group (8) that uses DHA as donor substrate and catalyzes the reversible formation of Fru-6-P from DHA and GAP (Fig. 1). Multiple sequence alignments of different Tal sequences demonstrate that FSA resides within the family of transaldolases (8, 9) and does show little similarity to DHAP-dependent aldolases, such as class I fructose-1,6-bisphosphate aldolase. Also the FSA monomer is highly similar to TalB exhibiting a similar (␤/␣) 8 -barrel fold (9). But in contrast to TalB, FSA forms a homo-decamer, which is arranged by two doughnut-shaped pentameric rings where the C-terminal helix of one subunit interacts with the adjacent subunit (9). With 220 amino acids compared with 317 amino acids FSA is smaller than TalB and shows about 20% sequence identity to TalB. Mechanistically, it is a class I aldolase, i.e. the reaction proceeds via a Schiff base intermediate.In this study, we addressed the differences b...

The majority of prokaryotic drugs are produced in glycosylated form, with the deoxygenation level in the sugar moiety having a profound influence on the drug's bioprofile. Chemical deoxygenation is challenging due to the need for tedious protective group manipulations. For a direct biocatalytic de novo generation of deoxysugars by carboligation, with regiocontrol over deoxygenation sites determined by the choice of enzyme and aldol components, we have investigated the substrate scope of the F178Y mutant of transaldolase B, TalB(F178Y), and fructose 6-phosphate aldolase, FSA, from E. coli against a panel of variously deoxygenated aldehydes and ketones as aldol acceptors and donors, respectively. Independent of substrate structure, both enzymes catalyze a stereospecific carboligation resulting in the D-threo configuration. In combination, these enzymes have allowed the preparation of a total of 22 out of 24 deoxygenated ketose-type products, many of which are inaccessible by available enzymes, from a [3×8] substrate matrix. Although aliphatic and hydroxylated aliphatic aldehydes were good substrates, D-lactaldehyde was found to be an inhibitor possibly as a consequence of inactive substrate binding to the catalytic Lys residue. A 1-hydroxy-2-alkanone moiety was identified as a common requirement for the donor substrate, whereas propanone and butanone were inactive. For reactions involving dihydroxypropanone, TalB(F178Y) proved to be the superior catalyst, whereas for reactions involving 1-hydroxybutanone, FSA is the only choice; for conversions using hydroxypropanone, both TalB(F178Y) and FSA are suitable. Structure-guided mutagenesis of Ser176 to Ala in the distant binding pocket of TalB(F178Y), in analogy with the FSA active site, further improved the acceptance of hydroxypropanone. Together, these catalysts are valuable new entries to an expanding toolbox of biocatalytic carboligation and complement each other well in their addressable constitutional space for the stereospecific preparation of deoxysugars.

SummaryIn Arabidopsis, the D-type cyclin CYCD3 is rate-limiting for transition of the G 1 /S boundary, and is transcriptionally upregulated at this point in cells re-entering the cell cycle in response to plant hormones and sucrose. However, little is known about the regulation of plant cell-cycle regulators at the protein level. We show here that CYCD3;1 is a phosphoprotein highly regulated at the level of protein abundance, whereas another D-type cyclin CYCD2;1 is not. The level of CYCD3;1 protein falls rapidly on sucrose depletion, correlated with the arrest of cells in G 1 phase, suggesting a rapid turnover of CYCD3;1. Treatment of exponentially growing cells with the protein synthesis inhibitor cycloheximide (CHX) con®rms that CYCD3;1 is normally a highly unstable protein, with a half-life of approximately 7 min on CHX treatment. In both sucrose-starved and exponentially growing cells, CYCD3;1 protein abundance increases in response to treatment with MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal), a reversible proteasome inhibitor, but not in response to the cysteine protease inhibitor E-64 or the calpain inhibitor ALLN (N-acetyl-leucyl-leucylnorleucinal). The increase on MG132 treatment is because of de novo protein synthesis coupled with the blocking of CYCD3;1 degradation. Longer MG132 treatment leads to C-terminal cleavage of CYCD3;1, accumulation of a hyperphosphorylated form and its subsequent disappearance. We conclude that CYCD3;1 is a highly unstable protein whose proteolysis is mediated by a proteasome-dependent pathway, and whose levels are highly dependent on the rate of CYCD3;1 protein synthesis.

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the first committed step in the biosynthesis of the bacterial cell wall component peptidoglycan. The enzyme is the target of the antibiotic fosfomycin. A lysine residue (K22), strictly conserved in MurAs and the structurally and mechanistically related 5-enolpyruvylshikimate 3-phosphate synthases (EPSPS), is located near the active center of the enzyme. This residue is thought to be involved directly in the binding of the substrate phosphoenolpyruvate (PEP) and also to participate in the conformational change leading to the formation of the catalytically competent enzyme complex. Using site-directed mutagenesis, we have replaced this lysine with arginine (K22R), valine (K22V), and glutamate (K22E). These mutant proteins were expressed, purified, and characterized in comparison to wild-type MurA and a previously described inactive C115S mutant protein. It was found that all three K22 mutant proteins had less than 0.5% of the wild-type activity. Using isothermal titration calorimetry, it could be shown that the binding parameters for the UDP-sugar nucleotide substrate are not affected by the mutations, except for the K22E mutant protein. Similarly, binding of PEP was found to be unaffected in the K22 mutant proteins as demonstrated by tryptophan fluorescence quench titrations. On the other hand, the level of formation of a covalent adduct with either PEP or fosfomycin with the thiol group of cysteine 115 was diminished. The propensity to form an adduct with PEP decreased in the following order: wild type >> K22R > K22V > K22E. A comparable effect was found on the formation of the inhibitory covalent adduct of MurA and the antibiotic fosfomycin. These results are discussed in terms of an involvement of lysine 22 in a conformational change of MurA.