The amino acid sequences of 301 glycosyl hydrolases and related enzymes have been compared. A total of 291 sequences corresponding to 39 EC entries could be classified into 35 families. Only ten sequences (less than 5% of the sample) could not be assigned to any family. With the sequences available for this analysis, 18 families were found to be monospecific (containing only one EC number) and 17 were found to be polyspecific (containing at least two EC numbers). Implications on the folding characteristics and mechanism of action of these enzymes and on the evolution of carbohydrate metabolism are discussed. With the steady increase in sequence and structural data, it is suggested that the enzyme classification system should perhaps be revised.

A classification of glycosyl hydrolases based on amino acid sequence similarities.

Dioxygen activation at mononuclear nonheme iron active sites: enzymes, models, and intermediates.

CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the raf family of protein kinases

We provide here a list of 221 P450 genes and 12 putative pseudogenes that have been characterized as of December 14, 1992. These genes have been described in 31 eukaryotes (including 11 mammalian and 3 plant species) and 11 prokaryotes. Of 36 gene families so far described, 12 families exist in all mammals examined to date. These 12 families comprise 22 mammalian subfamilies, of which 17 and 15 have been mapped in the human and mouse genome, respectively. To date, each subfamily appears to represent a cluster of tightly linked genes. This revision supersedes the previous updates [Nebert et al., DNA 6, 1–11, 1987; Nebert et al., DNA 8, 1–13, 1989; Nebert et al., DNA Cell Biol. 10, 1–14 (1991)] in which a nomenclature system, based on divergent evolution of the superfamily, has been described. For the gene and cDNA, we recommend that the italicized root symbol "CYP" for human ("Cyp" for mouse), representing "cytochrome P450," be followed by an Arabic number denoting the family, a letter designating...

https://www.liebertpub.com/doi/pdf/10.1089/dna.1993.12.1

The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature.

ion step follows the decarboxylation, which is consistent with the deuterium isotopic effects observed for thymine 7-hydroxylase which indicate that an irreversible step (or steps) occurs prior to the C-H bond breaking.395 It has also been shown for prolyl 4-hydroxylase that a substrate-derived radical is generated in the reaction, which is consistent with a rebound mechanism.437 It is important to point out that no oxygen intermediate (i.e., bridged superoxo or oxo-ferryl) has been observed for any R-KGdependent enzyme. This warrants future theoretical and experimental study. A detailed molecular mechanism has been proposed for IPNS based on spectroscopic and crystallographic studies.422 Resting IPNS/FeII is also 6C and thus relatively stable toward dioxygen. Substrate ACV binds directly to FeII IPNS through its thiolate group, providing an open coordination position at the FeII. O2 can then react to form an FeIII-superoxo intermediate. This intermediate is suggested422 to perform the first hydrogen-atom abstraction step and close the â-lactam ring, resulting in the formation of the first water molecule and generating an FeIVdO-II intermediate, which completes the second ringclosure process by hydrogen-atom abstraction forming a thiazolidine ring. Previously proposed mechanisms of ACCO involved direct binding of cosubstrate ascorbate to the iron before O2 as part of the oxygen activation process.438,439 The EPR and ESEEM studies of the NO complex of ACCO suggested a quite different molecular mechanism for ACCO.435 An FeIII-superoxo intermediate is proposed. Whether it is preceded by a 6C f 5C process with substrate binding is presently under study.440 This intermediate is thought to initiate a radical process by single hydrogen-atom abstraction or electron-coupled proton transfer (PT)ion or electron-coupled proton transfer (PT) from the bound amino group. The resulting substrate radical may undergo spontaneous conversion into products. The role of cosubstrate ascorbate is proposed to reduce the toxic peroxo byproduct to water. Alternatively, the two-electron reduction of FeIIIsuperoxo by the cosubstrate ascorbate could result in an FeIVdO-II intermediate which initiates the radical reaction.435 4. Rieske-Type Dioxygenases Biochemical Characterization. The Rieske ironsulfur center is a two iron-two sulfur cluster ([2Fe2S]) which has a 2His (on one iron), 2Cys (on the other iron) coordination environment, instead of the 4Cys present in plant ferredoxins. It plays a key role in the electron transport pathway in membranebound cytochrome complexes as well as in some dioxygenases.441 The latter are mainly comprised of two protein components: a reductase containing flavin and a ferredoxin [2Fe-2S], and a terminal oxygenase containing a Rieske [2Fe-2S] cluster and a non-heme iron active site.442 Except for the recently reported alkene monooxygenase that has a binuclear iron site in its terminal oxygenase,10 most of the Rieske-type oxygenases have a mononuclear iron site, which is believed to be the site of dioxygen activation and substrate oxygenation.442,443 The majority of the Rieske-type mononuclear non-heme oxygenases form a family of enzymes which are aromatic-ring-hydroxylating dioxygenases. These catalyze the regioand stereospecific cis-dihydroxylation of an aromatic ring using dioxygen and NAD(P)H (Table 1). Examples include benzene dioxygenase (BDO, EC 1.14.12.3),444 phthalate dioxygenase (PDO, EC 1.14.12.7),445 toluene dioxygenase (EC 1.14.12.11),446 and naphthalene 1,2-dioxygenase (NDO, EC 1.14.12.12),447 which initiate the aerobic degradation of aromatic compounds in the soil bacteria and are targets for bioengineering in bioremediation. This step is the first step in the pathway that ultimately leads to ring cleavage by the intraand extradiol dioxygenases (sections II.B.2 and II.C.1).443 Besides these bacterial dioxygenases, other Rieske-type mononuclear non-heme oxygenases include anthranilate 1,2-dioxygenase (EC 1.14.12.1),448 which deaminates and decarboxylates the substrate to produce catechol; chlorophenylacetate 3,4-dioxygenase (EC 1.14.2.13),449 which converts substrate to catechol with chloride elimination; and 4-methoxybenzoate O-demethylase (putidamonooxin),450 which catalyzes the conversion of 4-methoxybenzoic acid to 4-hydroxybenzoic acid and formaldehyde. The reductase component is usually a monomer (MW ) 12-15 kDa) and utilizes flavin to mediate ET from the two-electron donor NAD(P)H to the oneelectron acceptor [2Fe-2S] cluster and is specific to each terminal oxygenase; other electron donors do not support efficient oxygenation.442 The crystal structure of phthalate dioxygenase reductase is available.451 The terminal oxygenases are large protein aggregates (MW ) 150-200 kDa) containing either multiples of R subunits (BDO R2, PDO R4) or an equimolar combination of R and â subunits (toluene dioxygenase R2â2, NDO R3â3). The R subunits contain a Rieske [2Fe-2S] cluster and a catalytic non-heme FeII center. â subunits do not seem to be involved in the catalytic function (vide infra). Kinetics. Steady-state kinetic studies coupled with various rapid reaction studies of the partial reactions of PDO allowed Ballou et al. to propose a kinetic scheme (Scheme 15).443 On the basis of steady state 278 Chemical Reviews, 2000, Vol. 100, No. 1 Solomon et al.

Geometric and Electronic Structure/Function Correlations in Non-Heme Iron Enzymes

The ripening of avocado fruit is associated with the expression of a number of mRNAs concomitant with overt changes in texture and flavor. Two overlapping cDNAs for a mRNA that accumulates during ripening were identified. Sequence analysis of these two cDNAs revealed a polypeptide of 471 amino acids with characteristics of a typical P-450: an N-terminal hydrophobic membrane anchor, a conserved heme-binding domain in the C-terminal region, and patches of similarity to various P-450 family members. Further evidence that this polypeptide represents a cytochrome P-450 oxidase comes from the recent isolation and characterization of a cytochrome P-450 from ripe avocado mesocarp [O'Keefe, D. P. & Leto, K. J. (1989) Plant Physiol. 89, 1141-1149]. The N terminus of the predicted polypeptide in the cDNAs is identical to the N terminus of the purified avocado P-450. Gel blot analysis of RNA from fruit at various stages of ripening showed the accumulation of an 1800-nucleotide P-450 mRNA that hybridized to the P-450 cDNA. The P-450 protein predicted by the avocado cDNA sequence shares less than 40% positional identity with any known P-450 gene family. We propose therefore that it be placed in a separate family, P450LXXI, and that the corresponding gene from avocado be named cyp71A1.

https://www.pnas.org/content/pnas/87/10/3904.full.pdf

Sequence analysis of ripening-related cytochrome P-450 cDNAs from avocado fruit.

https://www.avocadosource.com/Journals/JBioChem/JBioChem_1992_267_5964-5967.pdf

Characterization and kinetic parameters of ethylene-forming enzyme from avocado fruit.

A cDNA library was constructed from poly(A)(+)RNA of ripe avocado fruit. Colony hybridization identified a number of ripening specific clones of which one, pAV5, was shown to be specific for cellulase. Hybrid selection with pAV5 provided a message from ripe fruit that on in vitro translation yielded a polypeptide of 53kD, comigrating with purified avocado cellulase on SDS polyacrylamide gel electrophoresis. The translation product was selectively immunoprecipitated by antiserum to purified avocado cellulase. Immunoblotting of unripe and ripe avocado fruit extracts following SDS-PAGE showed a plentiful immunoreactive polypeptide in ripe fruit, and essentially none in unripe fruit. Hybridization of pAV5 to poly(A)(+)-RNA from unripe and ripe avocado fruit demonstrated that there is at least a 50-fold increase in the cellulase message concentration during ripening. Thus, the expression of cellulase enzyme activity during ripening is regulated by the appearance of mRNA coding for cellulase rather than by either translational or post-translational control mechanisms.

Cellulase gene expression in ripening avocado fruit: the accumulation of cellulase mRNA and protein as demonstrated by cDNA hybridization and immunodetection

The final step of ethylene biosynthesis in plants is catalyzed by the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO). In addition to ACC, Fe(II), O2, CO2, and ascorbate are required for in vitro enzyme activity. Direct evidence for the role of the Fe(II) center in the recombinant avocado ACCO has now been obtained through formation of enzyme⋅(substrate or cofactor)⋅NO complexes. These NO adducts convert the normally EPR-silent ACCO complexes into EPR-active species with structural properties similar to those of the corresponding O2 complexes. It is shown here that the ternary Fe(II)ACCO⋅ACC⋅NO complex is readily formed, but no Fe(II)ACCO⋅ascorbate⋅NO complex could be observed, suggesting that ascorbate and NO are mutually exclusive in the active site. The binding modes of ACC and the structural analog alanine specifically labeled with 15N or 17O were examined by using Q-band electron nuclear double resonance (ENDOR). The data indicate that these molecules bind directly to the iron through both the α-amino and α-carboxylate groups. These observations are inconsistent with the currently favored mechanism for ACCO, in which it is proposed that both ascorbate and O2 bind to the iron as a step in O2 activation. We propose a different mechanism in which the iron serves instead to simultaneously bind ACC and O2, thereby fixing their relative orientations and promoting electron transfer between them to initiate catalysis.

Role of the nonheme Fe(II) center in the biosynthesis of the plant hormone ethylene

We present in this paper the structural analysis of two members of a small cellulase gene family, designated cel1 and cel2, from avocado. These genes were isolated by screening a λ EMBL3 genomic library with a ripening-induced cellulase cDNA. Restriction endonuclease and Southern blot analyses showed that the cel1 gene is highly homologous to the cellulase cDNA and thus represents a ripening-related cellulase gene. The other cellulase gene, cel2, is closely related to cel1, but is divergent at its 5′ end. The nucleotide sequence of a 5 kb region encompassing the cel1 gene was determined. Four previously characterized cellulase cDNAs from ripe fruit are identical to the eight exons of the cel1 gene. RNase protection and primer extension analyses were used to define the transcription start site of cel1 and to quantitate cel1 transcripts in ripening fruit. The cel1 mRNA was present at a low level in unripe fruit and increased 37-fold during ripening. Partial DNA sequence analysis of cel2 and comparison to the cel1 sequence revealed a high degree of similarity both at the DNA and deduced amino acid sequence levels. No characterized cellulase cDNAs derived from ripe fruit represent cel2 transcripts. These data suggest that the cel1 gene is responsible for a major portion, if not all, of the cellulase transcripts in ripe fruit. The DNA sequence of 1.4 kb of 5′ flanking DNA of the cel1 gene was compared to the upstream sequence of other ethylene-regulated genes. Several interesting upstream sequence motifs were identified and are discussed.

Rolf E. Christoffersen

Papers

Sequence analysis of ripening-related cytochrome P-450 cDNAs from avocado fruit.

Characterization and kinetic parameters of ethylene-forming enzyme from avocado fruit.

Cellulase gene expression in ripening avocado fruit: the accumulation of cellulase mRNA and protein as demonstrated by cDNA hybridization and immunodetection

Role of the nonheme Fe(II) center in the biosynthesis of the plant hormone ethylene

Isolation and characterization of a cellulase gene family member expressed during avocado fruit ripening.