C4 photosynthesis enables the capture of atmospheric CO2 and its concentration at the site of RuBisCO, thus counteracting the negative effects of low atmospheric levels of CO2 and high atmospheric levels of O2 (21 %) on photosynthesis. The evolution of this complex syndrome was a multistep process. It did not occur by simply recruiting pre-exiting components of the pathway from C3 ancestors which were already optimized for C4 function. Rather it involved modifications in the kinetics and regulatory properties of pre-existing isoforms of non-photosynthetic enzymes in C3 plants. Thus, biochemical studies aimed at elucidating the functional adaptations of these enzymes are central to the development of an integrative view of the C4 mechanism. In the present review, the most important biochemical approaches that we currently use to understand the evolution of the C4 isoforms of malic enzyme are summarized. It is expected that this information will help in the rational design of the best decarboxylation processes to provide CO2 for RuBisCO in engineering C3 species to perform C4 photosynthesis.

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The population genetic composition of Poa annua L. was studied by starch electrophoresis along a transect running NE from an organic reagents factory at Shanghai, China. Five enzyme systems were stained. We have reached the following preliminary conclusions: (1). Organic pollution has dramatically changed genotypic frequencies at some loci of Poa annua populations. At polluted sites, significant deviations from Hardy-Weinberg equilibrium were observed on loci Sod-1 and Me due to the excess of heterozygote. Especially in the two nearest sites to pollution source, all the individuals were heterozygous at locus Sod-1. The data suggests that heterozygotes were more tolerant to organic pollution than homozygotes, indicating the fitness superiority of heterozygotes. (2). A tendency towards clinal changes of allele frequencies was found at some polymorphic loci. Frequencies of the common alleles at loci Sod-1, Me and Fe-1 increased as the distance to the pollution source increased. (3). The effective number of alleles per locus, and the observed and expected heterozygosity were much higher in the pollution series than in the clear control site (Botanic Park population), but genetic multiplicity (number of alleles per locus) was lower than for the control. (4). Most genetic variability was found within populations, and only 2.56% were among populations of the polluted series. However, 9.48% of the total genetic variation occurred among populations when including the Botanic Park population. The genetic identity between populations of the pollution series (0.9869-1.0000, mean 0.9941) was higher than those between the pollution series and the Botanic Park population. UPGMA divided the five populations into two groups. One contained the four polluted populations, and the other only contained the Botanic Park population.

The citric acid or tricarboxylic acid cycle is a central element of higher-plant carbon metabolism which provides, among other things, electrons for oxidative phosphorylation in the inner mitochondrial membrane, intermediates for amino-acid biosynthesis, and oxaloacetate for gluconeogenesis from succinate derived from fatty acids via the glyoxylate cycle in glyoxysomes. The tricarboxylic acid cycle is a typical mitochondrial pathway and is widespread among alpha-proteobacteria, the group of eubacteria as defined under rRNA systematics from which mitochondria arose. Most of the enzymes of the tricarboxylic acid cycle are encoded in the nucleus in higher eukaryotes, and several have been previously shown to branch with their homologues from alpha-proteobacteria, indicating that the eukaryotic nuclear genes were acquired from the mitochondrial genome during the course of evolution. Here, we investigate the individual evolutionary histories of all of the enzymes of the tricarboxylic acid cycle and the glyoxylate cycle using protein maximum likelihood phylogenies, focusing on the evolutionary origin of the nuclear-encoded proteins in higher plants. The results indicate that about half of the proteins involved in this eukaryotic pathway are most similar to their alpha-proteobacterial homologues, whereas the remainder are most similar to eubacterial, but not specifically alpha-proteobacterial, homologues. A consideration of (a) the process of lateral gene transfer among free-living prokaryotes and (b) the mechanistics of endosymbiotic (symbiont-to-host) gene transfer reveals that it is unrealistic to expect all nuclear genes that were acquired from the alpha-proteobacterial ancestor of mitochondria to branch specifically with their homologues encoded in the genomes of contemporary alpha-proteobacteria. Rather, even if molecular phylogenetics were to work perfectly (which it does not), then some nuclear-encoded proteins that were acquired from the alpha-proteobacterial ancestor of mitochondria should, in phylogenetic trees, branch with homologues that are no longer found in most alpha-proteobacterial genomes, and some should reside on long branches that reveal affinity to eubacterial rather than archaebacterial homologues, but no particular affinity for any specific eubacterial donor.

Pigeon liver malic enzyme was inhibited by lutetium ion through a slow-binding process, which resulted in a concave down tracing of the enzyme activity assay. The fast initial rates were independent of lutetium ion concentration, while the slow steady-state rates decreased with increasing Lu(3+) concentration. The observed rate constant for the transition from initial rate to steady-state rate, k(obs), exhibited saturation kinetics as a function of Lu(3+) concentration, suggesting the involvement of an isomerization process between two enzyme forms (R-form and T-form). The binding affinity of Lu(3+) to the R-form is weaker (K(d,Lu) = 14 microM) than that of Mn(2+) (K(m,Mn) = 1.89 microM); however, Lu(3+) has much tighter binding affinity with the T-form ( = 0.83 microM). Lu(3+) was shown to be a competitive inhibitor with respect to Mn(2+), which suggests that Lu(3+) and Mn(2+) are competing for the same metal binding site of the enzyme. These observations are in accordance with the available crystal structure information, which shows a distorted active site region of the Lu(3+)-containing enzyme. Other divalent cations, i.e., Fe(2+), Cu(2+), or Zn(2+), also act as time-dependent slow inhibitors for malic enzyme. The dynamic quenching constants of the intrinsic fluorescence for the metal-free and Lu(3+)-containing enzymes are quite different, indicating the conformational differences between the two enzyme forms. The secondary structure of these two enzyme forms, on the other hand, was not changed. The above results indicated that replacement of the catalytically essential Mn(2+) by other metal ions leads to a slow conformational change of the enzyme and consequently alters the geometry of the active site. The transformed enzyme conformation, however, is unfavorable for catalysis. Both the chemical nature of the metal ion and its correct coordination in the active site are essential for catalysis.

The mechanism by which correctly folded proteins are recovered from stable complexes with groEL is not well understood. Certain target proteins require ATP and groES, while others seemingly dispense with the cochaperonin. Here, we examine the chaperonin-assisted folding of ribulose-1,5-bisphosphate carboxylase, malate dehydrogenase, and citrate synthase, three proteins that are believed to require both chaperonin components for successful reactivation. Surprisingly, in all cases, the need for groES depended on the folding environment. Under \"non-permissive\" conditions, where unassisted spontaneous folding could not occur, reactivation to the native state required the complete chaperonin system (e.g. groEL, groES, and MgATP). However, under \"permissive\" conditions where spontaneous folding could occur groES was no longer mandatory. Instead, upon the addition of ATP alone, all three target proteins could be released from groEL, in a form that was capable of reaching the native state. In the permissive setting, groES merely accelerated the rate of the ATP-dependent release process. The results suggest that the incompletely folded protein species that are released from groEL, in the absence of groES, are not necessarily committed to the native state. Similar to the unassisted folding reaction, they still partition between productive and unproductive folding pathways in an environment-dependent manner. It follows that the mechanistic contribution of the co-chaperonin, groES, and its physiological significance in cellular protein folding, could be entirely missed in a permissive in vitro environment.

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Halophilic malate dehydrogenase (hMDH) from Haloarcula marismortui has been isolated, purified and characterized by biochemical and biophysical solution studies. A stabilization mechanism at extremely high concentrations of salt, based on the formation of co-operative hydrate bonds between the protein and hydrated salt ions, was suggested from thermodynamic analysis of native enzyme solutions. Recently the gene coding for hMDH was isolated and sequenced and an active enzyme cloned (F. Cendrin, J. Chroboczek, G. Zaccai, H. Eisenberg and M. Mevarech, unpublished work). A study of the crystal structure of hMDH in a high-salt physiological medium is in progress (O. Butbul-Dym & J. Sussman, personal communication). Here we discuss in depth implications of these recent developments on our earlier results.