We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.

Metagenomics has made accessible an enormous reserve of global biochemical diversity. To tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. We have validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalytic residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.

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Glycerol 3-phosphate cytidylyltransferase from Staphylococcus aureus (TarD Sa ) has been expressed in Escherichia coli, puri®ed to homogeneity and crystallized. The strategy used for determining crystallization conditions employed hanging-drop sparse-matrix screens and required a combination of three different optimization approaches. Speci®cally, the presence or absence of cofactors needed to be surveyed, the effects of small-molecule additives required exploration and the rate of vapour-diffusion had to be varied in order to obtain crystals of TarD Sa suitable for diffraction studies. Crystals thus obtained belong to the space group P3 1 21, with unit-cell parameters a = b = 92.2, c = 156.1 A Ê , and contain four TarD Sa molecules per asymmetric unit. The resolution limit observed for these crystals using synchrotron radiation is 3.0 A Ê .

SummaryGene sequences annotated as proteins of unknown or non‐specific function and hypothetical proteins account for a large fraction of most genomes. In the strictly anaerobic and organohalide respiring D ehalococcoides mccartyi, this lack of annotation plagues almost half the genome. Using a combination of bioinformatics analyses and genome‐wide metabolic modelling, new or more specific annotations were proposed for about 80 of these poorly annotated genes in previous investigations of D . mccartyi metabolism. Herein, we report the experimental validation of the proposed reannotations for two such genes (KB1_0495 and KB1_0553) from D . mccartyi strains in the KB‐1 community. KB1_0495 or DmIDH was originally annotated as an NAD +‐dependent isocitrate dehydrogenase, but biochemical assays revealed its activity primarily with NADP + as a cofactor. KB1_0553, also denoted as DmPMI, was originally annotated as a hypothetical protein/sugar isomerase domain protein. We previously proposed that it was a bifunctional phosphoglucose isomerase/phosphomannose isomerase, but only phosphomannose isomerase activity was identified and confirmed experimentally. Further bioinformatics analyses of these two protein sequences suggest their affiliation to potentially novel enzyme families within their respective larger enzyme super families.