Koichi Nonaka

An ATP-independent strategy for amide bond formation in antibiotic biosynthesis.

A-503083 B, a capuramycin-type antibiotic, contains an L-aminocaprolactam and an unsaturated hexuronic acid that are linked via an amide bond. A putative class C beta-lactamase (CapW) was identified within the biosynthetic gene cluster that-in contrast to the expected beta-lactamase activity-catalyzed an amide-ester exchange reaction to eliminate the L-aminocaprolactam with concomitant generation of a small but significant amount of the glyceryl ester derivative of A-503083 B, suggesting a potential role for an ester intermediate in the biosynthesis of capuramycins. A carboxyl methyltransferase, CapS, was subsequently demonstrated to function as an S-adenosylmethionine-dependent carboxyl methyltransferase to form the methyl ester derivative of A-503083 B. In the presence of free L-aminocaprolactam, CapW efficiently converts the methyl ester to A-503083 B, thereby generating a new amide bond. This ATP-independent amide bond formation using methyl esterification followed by an ester-amide exchange reaction represents an alternative to known strategies of amide bond formation.

Efficient Antibody Production upon Suppression of O Mannosylation in the Yeast Ogataea minuta

ABSTRACT When antibodies were expressed in the methylotrophic yeast Ogataea minuta, we found that abnormal O mannosylation occurred in the secreted antibody. Yeast-specific O mannosylation is initiated by the addition of mannose at serine (Ser) or threonine (Thr) residues in the endoplasmic reticulum via protein O mannosyltransferase (Pmt) activity. To suppress the addition of O-linked sugar chains on antibodies, we examined the possibility of inhibiting Pmt activity by the addition of a Pmt inhibitor during cultivation. The Pmt inhibitor was found to partially suppress the O mannosylation on the antibodies. Surprisingly, the suppression of O mannosylation was associated with an increased amount of assembled antibody (H2L2) and enhanced the antigen-binding activity of the secreted antibody. In this study, we demonstrated the expression of human antibody in O. minuta and elucidated the relationship between O mannosylation and antibody production in yeast.

The Biosynthesis of Liposidomycin-like A-90289 Antibiotics Featuring a New Type of Sulfotransferase

Enzymes involved in peptidoglycan cell wall biosynthesis are proven targets for antibacterial drugs that have revolutionized medicine. Remarkably, however, the majority of the enzymes involved in peptidoglycan biosynthesis have yet to be exploited in the clinic. Bacterial translocase I (also annotated as MraY), which initiates the lipid cycle of peptidoglycan cell wall biosynthesis by transfer of phospho-N-acetylmuramic acid-pentapeptide from UDP-N-acetylmuramic acid-pentapeptide to undecaprenyl phosphate, represents one such enzyme for which there are no marketed antibiotic drugs. Given the emergence and re-emergence of drug-resistance pathogens, the essential nature of peptidoglycan in the viability of bacteria, and the lack of a bacterial translocase I activity in mammals, MraY is an attractive target for the design and development of new antibiotics. Recent efforts in several laboratories have revealed that several structurally diverse natural products potently inhibit bacterial translocase I. This includes several families of nucleoside antibiotics that have unique chemical scaffolds relative to clinically used antibiotics. The liposidomycins belong to one such family of nucleoside antibiotics called fatty acyl nucleosides that also includes the caprazamycins isolated from Streptomyces sp. MK730-62F2. The liposidomycins, the structure of which was initially reported in 1988, are characterized by four moieties, a 5’-C-glycyluridine, a 2’-sulfated aminoribose, a diazepanone, and a b-hydroxy fatty acid moiety of variable carbon chain length modified with an unusual 3-methylglutaryl group at the b-position. Caprazamycins, in contrast to liposidomycins, contain an additional permethylated rhamnose and lack the 2’-O-sulfate moiety. During screening for new compounds that inhibit bacterial translocase I, we isolated a series of related compounds from Streptomyces sp. SANK 60405 termed A-90289s that had properties characteristic of the fatty acyl nucleoside antibiotics including variable fatty acid side chains. NMR analysis—including DQF COSY, HMBC, and NOESY experiments—and MS analysis with collision-induced dissociation (CID) were consistent with A-90289A, consisting of a b-hydroxy palmitic acid moiety and having a structure identical to that of caprazamycin A but also containing a sulfate group characteristic of liposidomycins B-I and the other liposidomycins (Scheme 1). However, in contrast

The Biosynthesis of Capuramycin-type Antibiotics IDENTIFICATION OF THE A-102395 BIOSYNTHETIC GENE CLUSTER, MECHANISM OF SELF-RESISTANCE, AND FORMATION OF URIDINE-5′-CARBOXAMIDE

Background: Several nucleoside antibiotics contain a uridine-5′-carboxamide core of unclear origin. Results: The A-102395 biosynthetic gene cluster was cloned, a genetic system was developed, and three enzymes were characterized in vivo and in vitro. Conclusion: Uridine-5′-carboxamide originates from UMP and l-Thr by sequential reactions catalyzed by a dioxygenase and transaldolase. Significance: The results provide the first opportunity to methodically interrogate the biosynthesis of these unusual antibiotics. A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discovered using a screen to identify inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan cell wall biosynthesis. Like the parent capuramycin, A-500359s and A-503083s consist of three structural components: a uridine-5′-carboxamide (CarU), a rare unsaturated hexuronic acid, and an aminocaprolactam, the last of which is substituted by an unusual arylamine-containing polyamide in A-102395. The biosynthetic gene clusters for A-500359s and A-503083s have been reported, and two genes encoding a putative non-heme Fe(II)-dependent α-ketoglutarate:UMP dioxygenase and an l-Thr:uridine-5′-aldehyde transaldolase were uncovered, suggesting that C–C bond formation during assembly of the high carbon (C6) sugar backbone of CarU proceeds from the precursors UMP and l-Thr to form 5′-C-glycyluridine (C7) as a biosynthetic intermediate. Here, isotopic enrichment studies with the producer of A-503083s were used to indeed establish l-Thr as the direct source of the carboxamide of CarU. With this knowledge, the A-102395 gene cluster was subsequently cloned and characterized. A genetic system in the A-102395-producing strain was developed, permitting the inactivation of several genes, including those encoding the dioxygenase (cpr19) and transaldolase (cpr25), which abolished the production of A-102395, thus confirming their role in biosynthesis. Heterologous production of recombinant Cpr19 and CapK, the transaldolase homolog involved in A-503083 biosynthesis, confirmed their expected function. Finally, a phosphotransferase (Cpr17) conferring self-resistance was functionally characterized. The results provide the opportunity to use comparative genomics along with in vivo and in vitro approaches to probe the biosynthetic mechanism of these intriguing structures.

Evidence that oxidative dephosphorylation by the nonheme Fe(II), α‐ketoglutarate:UMP oxygenase occurs by stereospecific hydroxylation

LipL and Cpr19 are nonheme, mononuclear Fe(II)‐dependent, α‐ketoglutarate (αKG):UMP oxygenases that catalyze the formation of CO2, succinate, phosphate, and uridine‐5′‐aldehyde, the last of which is a biosynthetic precursor for several nucleoside antibiotics that inhibit bacterial translocase I (MraY). To better understand the chemistry underlying this unusual oxidative dephosphorylation and establish a mechanistic framework for LipL and Cpr19, we report herein the synthesis of two biochemical probes—[1′,3′,4′,5′,5′‐2H]UMP and the phosphonate derivative of UMP—and their activity with both enzymes. The results are consistent with a reaction coordinate that proceeds through the loss of one 2H atom of [1′,3′,4′,5′,5′‐2H]UMP and stereospecific hydroxylation geminal to the phosphoester to form a cryptic intermediate, (5′R)‐5′‐hydroxy‐UMP. Thus, these enzyme catalysts can additionally be assigned as UMP hydroxylase‐phospholyases.

Choosing the right protein A affinity chromatography media can remove aggregates efficiently

Protein A chromatography (PAC) is commonly used as an efficient capture step in monoclonal antibody (mAb) separation processes. Usually dynamic binding capacity is used for choosing the right PAC. However, if aggregates can be efficiently removed during elution, it can make the following polishing steps easier. In this study a method for choosing the right PAC media in terms of mAb aggregate removal is proposed. Linear pH gradient elution experiments of two different mAbs on various PAC columns are carried out, where the elution behavior of aggregates as well as the monomer is measured. Aggregates of one mAb are more strongly retained compared with the mAb monomer. Another mAb showed different elution behavior, where the aggregates are eluted as both the weakly and strongly retained peaks. In order to remove the two types of aggregates by stepwise elution two protocols are tested. The first protocol A consisted of the sample loading, the wash with the equilibration buffer and the low pH elution. The wash stage of the second protocol B included the wash with 1.0 M arginine. No detectable peaks are observed during the wash stage of protocol A whereas significant peaks are monitored during the arginine wash of protocol B. One of the PAC columns showed a smaller peak during the arginine wash. In addition, both aggregate removal and monomer yield are higher with protocol B compared with the other PAC columns. This method is found to be useful for choosing the right PAC column.

Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry

To screen a high-producing recombinant Chinese hamster ovary (CHO) cell from transfected cells is generally laborious and time-consuming. We developed an efficient enrichment strategy for high-producing cell screening using flow cytometry (FCM). A stable pool that had possibly shown a huge variety of monoclonal antibody (mAb) expression levels was prepared by transfection of an expression vector for mAb production to a CHO cell. To enrich high-producing cells derived from a stable pool stained with a fluorescent-labeled antibody that binds to mAb presented on the cell surface, we set the cell size and intracellular density gates based on forward scatter (FSC) and side scatter (SSC), and collected the brightest 5% of fluorescein isothiocyanate (FITC)-positive cells from each group by FCM. The final product concentration in a fed-batch culture of cells sorted without FSC and SSC gates was 1.2-1.3-times higher than that of unsorted cells, whereas that of cells gated by FSC and SSC was 3.4-4.7-fold higher than unsorted cells. Surprisingly, the fraction with the highest final product concentration indicated the smallest value of FSC and SSC, and the middle value of fluorescence intensity among all fractionated cells. Our results showed that our new screening strategy by FCM based on FSC and SSC gates could achieve an efficient enrichment of high-producing cells with the smallest value of FSC and SSC.

The Role of a Nonribosomal Peptide Synthetase in l-Lysine Lactamization During Capuramycin Biosynthesis.

Capuramycins are one of several known classes of natural products that contain an l‐Lys‐derived l‐α‐amino‐ɛ‐caprolactam (l‐ACL) unit. The α‐amino group of l‐ACL in a capuramycin is linked to an unsaturated hexuronic acid component through an amide bond that was previously shown to originate by an ATP‐independent enzymatic route. With the aid of a combined in vivo and in vitro approach, a predicted tridomain nonribosomal peptide synthetase CapU is functionally characterized here as the ATP‐dependent amide‐bond‐forming catalyst responsible for the biosynthesis of the remaining amide bond present in l‐ACL. The results are consistent with the adenylation domain of CapU as the essential catalytic component for l‐Lys activation and thioesterification of the adjacent thiolation domain. However, in contrast to expectations, lactamization does not require any additional domains or proteins and is likely a nonenzymatic event. The results set the stage for examining whether a similar NRPS‐mediated mechanism is employed in the biosynthesis of other l‐ACL‐containing natural products and, just as intriguingly, how spontaneous lactamization is avoided in the numerous NRPS‐derived peptides that contain an unmodified l‐Lys residue.