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Synechococcus cyanobacteria are widespread in the marine environment, as the extensive pigment diversity within their light-harvesting phycobilisomes enables them to utilize various wavelengths of light for photosynthesis. The phycobilisomes of Synechococcus sp. RS9916 contain two forms of the protein phycoerythrin (PEI and PEII), each binding two chromophores, green-light absorbing phycoerythrobilin and blue-light absorbing phycourobilin. These chromophores are ligated to specific cysteines via bilin lyases, and some of these enzymes, called lyase isomerases, attach phycoerythrobilin and simultaneously isomerize it to phycourobilin. MpeV is a putative lyase isomerase whose role in PEI and PEII biosynthesis is not clear. We examined MpeV in RS9916 using recombinant protein expression, absorbance spectroscopy, and tandem mass spectrometry. Our results show that MpeV is the lyase isomerase that covalently attaches a doubly linked phycourobilin to two cysteine residues (C50, C61) on the β-subunit of both PEI (CpeB) and PEII (MpeB). MpeV activity requires that CpeB or MpeB is first chromophorylated by the lyase CpeS (which adds phycoerythrobilin to C82). Its activity is further enhanced by CpeZ (a homolog of a chaperone-like protein first characterized in Fremyella diplosiphon). MpeV showed no detectable activity on the α-subunits of PEI or PEII. The mechanism by which MpeV links the A and D rings of phycourobilin to C50 and C61 of CpeB was also explored using site-directed mutants, revealing that linkage at the A ring to C50 is a critical step in chromophore attachment, isomerization, and stability. These data provide novel insights into β-PE biosynthesis and advance our understanding of the mechanisms guiding lyase isomerases., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Class II diterpene cyclases catalyze bicyclization of geranylgeranyl diphosphate. While this reaction typically is terminated via methyl deprotonation to yield copalyl diphosphate, in rare cases hydroxylated bicycles are produced instead. Abietadiene synthase is a bifunctional diterpene cyclase that usually produces a copalyl diphosphate intermediate. Here it is shown that substitution of aspartate for a conserved histidine in the class II active site of abietadiene synthase leads to selective production of 8α-hydroxy-CPP instead, demonstrating striking plasticity.

With more than 55,000 members identified so far in all forms of life, the family of terpene or terpenoid natural products represents the epitome of molecular biodiversity. A well-known and important member of this family is the polycyclic diterpenoid Taxol (paclitaxel), which promotes tubulin polymerization and shows remarkable efficacy in cancer chemotherapy. The first committed step of Taxol biosynthesis in the Pacific yew (Taxus brevifolia) is the cyclization of the linear isoprenoid substrate geranylgeranyl diphosphate (GGPP) to form taxa-4(5),11(12)diene, which is catalysed by taxadiene synthase. The full-length form of this diterpene cyclase contains 862 residues, but a roughly 80-residue amino-terminal transit sequence is cleaved on maturation in plastids. We now report the X-ray crystal structure of a truncation variant lacking the transit sequence and an additional 27 residues at the N terminus, hereafter designated TXS. Specifically, we have determined structures of TXS complexed with 13-aza-13,14-dihydrocopalyl diphosphate (1.82 Å resolution) and 2-fluorogeranylgeranyl diphosphate (2.25 Å resolution). The TXS structure reveals a modular assembly of three α-helical domains. The carboxy-terminal catalytic domain is a class I terpenoid cyclase, which binds and activates substrate GGPP with a three-metal ion cluster. The N-terminal domain and a third 'insertion' domain together adopt the fold of a vestigial class II terpenoid cyclase. A class II cyclase activates the isoprenoid substrate by protonation instead of ionization, and the TXS structure reveals a definitive connection between the two distinct cyclase classes in the evolution of terpenoid biosynthesis.

Enzyme function is much less conserved than anticipated, i.e., the requirement for sequence similarity that implies similarity in enzymatic function is much higher than the requirement that implies similarity in protein structure. This is because the function of an enzyme is an extremely complicated problem that may involve very subtle structural details as well as many other physical chemistry factors. Accordingly, if simply based on the sequence similarity approach, it would hardly get a decent success rate in predicting enzyme sub-class even for a dataset consisting of samples with 50% sequence identity. To cope with such a situation, the GO-PseAA predictor was adopted to identify the sub-class for each of the six main enzyme families. It has been observed that, even for the much more stringent datasets in which none of the enzymes has 25% sequence identity to any others, the overall success rates are 73-95%, suggesting that the GO-PseAA predictor can catch the core features of the statistical samples concerned and may become a useful high throughput tool in proteomics and bioinformatics.

Using a combination of several methods for protein sequence comparison and motif analysis, it is shown that the four recently described pseudouridine syntheses with different specificities belong to four distinct families. Three of these families share two conserved motifs that are likely to be directly involved in catalysis. One of these motifs is detected also in two other families of enzymes that specifically bind uridine, namely deoxycitidine triphosphate deaminases and deoxyuridine triphosphatases. It is proposed that this motif is an essential part of the uridine-binding site. Two of the pseudouridine syntheses, one of which modifies the anticodon arm of tRNAs and the other is predicted to modify a portion of the large ribosomal subunit RNA belonging to the peptidyltransferase center, are encoded in all extensively sequenced genomes, including the 'minimal' genome of Mycoplasma genitalium. These particular RNA modifications and the respective enzymes are likely to be essential for the functioning of any cell.

Sequencing of N-terminal and internal peptide fragments of the purified 17 kDa Bacillus subtilis peptidyl-prolyl cis-trans isomerase (PPIase) revealed sequence identity to conserved regions of a number of eukaryotic and prokaryotic cyclophilins. Using two oligonucleotide primers corresponding to the N-terminus and a highly conserved internal amino acid sequence, polymerase chain reactions (PCR) with B. subtilis genomic DNA were carried out. The resultant PCR fragment of 335 bp was cloned, sequenced and subsequently used as a probe for screening a lambda Zap II gene library of B. subtilis. Two overlapping positive clones of 5 and 7 kb containing the B. subtilis PPIase gene (ppiB), which is 432 bp in length and encodes a protein of 144 amino acid residues, were identified and two distinct transcriptional initiation sites at the 5' end of ppiB were mapped. The entire region (35 kb) between spoVA and serA was recently sequenced in B. subtilis, and an open reading frame (ORF) that encodes a putative peptidyl-prolyl cis-trans isomerase at about 210 degrees on the B. subtilis genetic map was located. This putative PPIase is identical to PPiB. We have overexpressed the ppiB gene in Escherichia coli, purified the encoded protein to apparent homology and shown that it exhibits PPIase activity. In addition, the recombinant PPiB shows a significant inhibition of PPIase activity by cyclosporin A (CsA) at a level comparable to that observed for the B. subtilis enzyme. Interestingly the B. subtilis PPIase shows about 40% identity to eukaryotic PPIases and less similarity to those of Gram-negative bacteria (27-32% identity). Like other interruption mutants of yeast and Neurospora, which lack a functional cyclophilin gene, a B. subtilis mutant containing ppiB::cat, a cat-interrupted copy of ppiB in the chromosome, is viable.

Candida tropicalis peroxisomes contain a 905-residue trifunctional enzyme with hydratase-dehydrogenase-epimerase activity that is important in fatty acid beta-oxidation. At its amino terminus are two tandem copies of an approximately 280 residue domain of unknown function. We provide evidence that this domain is homologous to oxidoreductases used for metabolizing sugars and synthesizing antibiotics and steroids such as estradiol, androstenedione, corticosterone, and hydrocortisone. The trifunctional enzyme shows no sequence similarity to the bifunctional hydratase-dehydrogenase found in animal peroxisomes and plant glyoxysomes, which are homologs of each other. We suggest that the C. tropicalis trifunctional enzyme and the animal and plant bifunctional enzymes have different ancestors.

Recommendations for the nomenclature and coding of enzymes as presented by the International Union of Pure and Applied Chemistry and the International Union of Biochemistry are summarized and discussed. Units for reporting catalytic concentration of enzymes are briefly reviewed and abbreviations that have been proposed for enzyme names are also described.